Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures.

Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures. Carlos López Ramón y Cajal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6185543/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The discovery of Novel Structured Entities (NSE) in meteorite-derived cultures provides an opportunity to investigate structured formations with unique physicochemical properties and their potential role in the stabilization of genetic elements. This study employs Live Optical LED microscopy to characterize and dynamically visualize the structural properties and organizational stages of NSE under controlled conditions. Meteorite fragments from diverse locations and compositions were cultured in Dulbecco’s Modified Eagle’s Medium or sterile distilled water, enabling the observation of NSE morphologies, including motile vesicular structures (Free Forms), protective biofilms (Biofilm Forms), and highly mineralized laminated fibers (Resistant Forms). Staining with MTG revealed fluorescence across all observed NSE stages, suggesting an interaction with specific structural components rather than direct metabolic activity. Our findings indicate that these structured formations emerge in meteorite-derived cultures and exhibit properties suggesting resilience under extreme environmental conditions. While their organized morphology and co-occurrence with ssDNA sequences are intriguing, further research is required to determine whether this association holds any functional significance or results from unrelated culture dynamics. These results provide a structural framework for a parallel genomic study, which detected previously uncharacterized single-stranded DNA (ssDNA) sequences within the same cultures, warranting further investigation into potential associations. While the origins and nature of these structured formations remain to be fully elucidated, these findings underscore the importance of integrating molecular, structural, and functional analyses to explore the persistence of genetic elements in extreme environments and their potential implications for prebiotic evolution. Astrobiology Evolutionary Biology Meteoritics Astrochemistry Novel Structured Entities (NSE) Meteorite Cultures Live Optical LED (LOL) Microscopy Astrobiology Microscopy ssDNA Persistence Prebiotic Evolution Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Introduction In biomedicine, the ongoing development of innovative methodologies has significantly enhanced the study of biological samples, leading to the discovery of novel structures and a deeper understanding of biological processes. These advancements underscore the necessity for continuous refinement of observational techniques to ensure the precise characterization of biological entities in their native state, capturing their morphology, dynamics, and functional properties while minimizing observational artifacts. In this study, we present an innovative microscopy approach—Live Optical LED (LOL)—designed for real-time visualization of biological structures in conditions that closely replicate their native environment. Unlike conventional histological techniques, which often require staining and may introduce artifacts, LOL allows direct observation without chemical modifications. This method enhances the detection of dynamic structures that could otherwise remain undetected due to sample processing limitations. The LOL technique operates exclusively with LED illumination, which provides superior light quality, precise wavelength control, and instantaneous activation. Unlike halogen-based systems, LED illumination, when integrated with an RGB camera, enables the acquisition of high-resolution, color-accurate images without requiring additional filtering or post-processing. This configuration ensures the preservation of the structural integrity and native characteristics of the samples. Attempts to achieve similar results with alternative light sources were unsuccessful, emphasizing the critical role of LED technology in the LOL methodology. This advanced configuration, combined with an integrated RGB camera, allows for immediate analysis of biologically active samples in real time, representing a significant advancement in the study of living tissues and biological systems (Aswani, 2016 ). Using the LOL methodology, we examined hundreds of freshly obtained human amniotic membranes immediately after labor, mounting them on microscope slides without fixation or staining. During these observations, we identified an uncharacterized structure interacting with both the epithelial layer and deeper regions of the amniotic membrane (Fig. 1 ). Initially termed the New Biological Structure (NBS), this formation exhibited distinctive features suggestive of a biological origin and was consistently observed across samples. In addition, it showed positive staining with MitoTracker™ Green FM (MTG), a marker typically associated with bioenergetic activity, reinforcing its potential biological significance. This discovery prompted questions regarding its prevalence in both biological and non-biological contexts. Previous studies initially employed the term New Biological Structure (NBS) to describe complex morphologies observed in meteorite-derived cultures (López Ramón y Cajal, 2025). However, subsequent analyses highlighted critical uncertainties regarding the strictly biological nature of these structures. To reflect a more accurate and cautious interpretation, we now propose the term Novel Structured Entities (NSE). The NSE concept deliberately emphasizes their dynamic structural organization without presupposing biological activity or origin. Indeed, NSE could represent highly organized assemblies emerging from purely physicochemical or prebiotic self-organization processes. Although their coexistence with genetic elements is intriguing, we stress clearly that, at present, no direct functional relationship or biological origin has been confirmed. Thus, their precise nature—biological, prebiotic, or purely physicochemical—remains open, and our study should be viewed as foundational and exploratory, inviting further experimental scrutiny and validation. Previous metagenomic analyses of the same meteorite-derived cultures identified ssDNA sequences that lack homologs in current databases (López Ramón y Cajal, 2025) These sequences contain conserved structural motifs, raising questions about their origin and potential significance. Although their presence in the same cultures as NSE is an interesting observation, there is currently no evidence indicating a direct interaction or functional relationship between them. Further experimental validation is necessary to assess whether this correlation is biologically meaningful or merely a coincidental occurrence within the culture conditions. To further investigate this hypothesis, we examined NSE across a range of sources—including human blood smears (Figs. 2 and 3 ), sterile plant fibers, biogenic minerals (e.g., magnetite and siderite), and notably, cultures derived from meteorite fragments. The meteorite-derived cultures were also analyzed in a parallel genomic study (López Ramón y Cajal, 2025), which confirmed an association between NSE and previously uncharacterized ssDNA sequences under identical culture conditions, thereby establishing a direct link between the structural and genetic investigations of these entities. The observation of NSE in meteorite-derived cultures prompts questions about their structural organization and potential stability under laboratory-simulated extreme conditions. Although their coexistence with biogenic minerals such as magnetite and siderite—minerals often associated with biological processes (Chaudhuri, Lack, & Coates, 2001) —is intriguing, we emphasize that it remains unclear whether these associations are meaningful or merely coincidental. While previous studies have suggested that biogenic minerals might contribute to structural stabilization in complex environments (Van Cappellen, 2003), further research is necessary to determine if NSE are actively involved in such stabilization processes or simply occur alongside these minerals. Likewise, the correlation observed between NSE and novel ssDNA sequences within these cultures invites further exploration; however, no direct evidence currently indicates a functional or causal relationship between them. Thus, additional rigorous experimental studies are required to clarify the precise nature of these interactions. This study aims to provide a detailed microscopic characterization and dynamic visualization of NSE, focusing on their detection, structural organization, and potential adaptability within meteorite-derived cultures. By utilizing the same cultures analyzed in the parallel genomic study (López Ramón y Cajal, 2025), this research complements molecular findings with high-resolution imaging, forming a cohesive framework for understanding the structural and genetic properties of NSE. Ultimately, our efforts aim to provide a detailed description and better understanding of the structural characteristics and potential adaptability of NSE observed under laboratory conditions simulating extreme environments. Although the observation of these structured entities under simulated extreme or extraterrestrial-like conditions is provocative, we explicitly underline that their exact nature remains uncertain. Our data alone cannot confirm whether NSE originate from biological, prebiotic, or purely physicochemical processes. Consequently, the interpretations presented here are preliminary and intended to stimulate rigorous, follow-up experimental studies rather than to support definitive conclusions about their biological significance or extraterrestrial origin. Future research should aim explicitly at elucidating the physicochemical conditions, molecular mechanisms, and possible functional implications underlying NSE formation and persistence. Material and methods The descriptive study of the NBS was conducted by the author at the Álvaro Cunqueiro Hospital in Vigo, serving as the basis for genomic analysis. The research project on the NSE was approved by the Ethics Committee for Research of Pontevedra-Vigo-Orense (Registration Codes: 2019/151 and 2019/648). Culturing of meteorite samples Meteorite fragments evaluated in the descriptive study of the NSE were cultured under the same controlled conditions and methodologies as described in the genomic study preprint (López Ramón y Cajal, 2025). The cultures were performed in sterile Nunc™ EasYFlask™ 25 cm² flasks with Nunclon™ Delta Surface, ensuring an optimal environment for potential growth. Two distinct culture media were employed to assess the adaptability and growth potential of biological entities within the samples. These protocols were meticulously designed to maintain a sterile and controlled environment, ensuring the reliability of subsequent DNA extraction and analysis of potential biological entities associated with the meteorite samples. Negative controls and validation procedures. To ensure methodological rigor and exclude possible contamination or experimental artifacts, rigorous negative controls were implemented at key experimental steps. These controls included culturing sterile media (DMEM and sterile distilled water) without meteorite samples under identical experimental conditions (temperature, shaking, and light exposure) to evaluate the spontaneous emergence of structures. Additionally, environmental controls (culture flasks briefly exposed to laboratory air without meteorites) were cultured to rule out airborne contamination. DNA extraction was performed on these negative controls using identical protocols, confirming no detectable DNA contaminants. Furthermore, independent replicates involving meteorites of diverse origins consistently yielded NSE, whereas none appeared in the negative controls, reinforcing the reliability and specificity of our findings. Additionally, the reproducibility of these results was confirmed through independent replicates, using meteorites of varying origins. Preparation of samples for cultures and culture considerations. We cultured one or two fragments of meteorites, each measuring 5–10 mm (< 2 cm) per culture sample, following an exhaustive cleaning and decontamination process. The procedure involved treatment with didecyldimethylammonium chloride and non-ionic surfactants for 30 minutes, followed by immersion in pure bleach (5% sodium hypochlorite) for an additional 30 minutes and thorough rinsing with sterile water. After this process, meteorite samples were cultured under controlled laboratory conditions. Meteorites used. A total of seven meteorites were utilized in the study, providing a diverse range of compositions and origins (Fig. 4 ). The details of each meteorite are as follows: Meteorite 1 (AIQUILE): Type: ordinary chondrite H5. Fall location: Cochabamba, Bolivia. Confirmed fall: November 20, 2016. Status: listed in the Meteoritical Bulletin Database. Meteorite 2 (NWA 781): Type: ordinary chondrite LL6. Fall location: Morocco. Fall year: 2001. Status: listed in the Meteoritical Bulletin Database. Meteorite 3 (Chelyabinsk): Type: ordinary chondrite LL5. Fall location: Chelyabinskaya oblast’, Russia. Confirmed fall: February 15, 2013. Status: listed in the Meteoritical Bulletin Database. Meteorite 4 (NWA 803): Type: ordinary chondrite L6. Fall location: Northwest Africa—Morocco. Fall year: 2001. Status: listed in the Meteoritical Bulletin Database. Meteorite 5 (NWA): Type: achondrite from the eucrite group. Fall location: Northwest Africa—Algeria. Fall year: 2021. Status: not listed in the Meteoritical Bulletin Database. Meteorite 6 (NWA 869): Type: ordinary chondrite L4-6. Fall location: Northwest Africa—Algeria. Fall year: 2000. Status: listed in the Meteoritical Bulletin Database. Meteorite 7 (Northwest Africa, NWA 12455): Type: carbonaceous chondrite CR7. Fall location: Northwest Africa. Fall year: 2018. Status: listed in the Meteoritical Bulletin Database. The meteorite samples were selected based on their compositional richness, prioritizing ordinary chondrites and carbonaceous chondrites due to their diverse mineral and organic content. These classifications provided an optimal framework for exploring potential biological or prebiotic signatures, as they are known to harbor complex organic molecules and support prebiotic chemistry (Sephton, 2002). Culturing procedure. Cultures were performed at room temperature (22–23°C). Meteorite fragments were placed into flasks containing the culture medium, and the flasks were vigorously shaken to facilitate the fracturing of the meteorite fragments and the release of potential internal contents. This shaking process was repeated daily at 07:00 to ensure consistency. Additionally, the cultures were evaluated at least twice daily through a detailed examination of the flasks using an inverted microscope to identify any potential forms of the NSE. These systematic evaluations aimed to detect and document the emergence of NSE structures as early as possible in the culture process. Two distinct culture media were utilized (20 mL per sterile Nunc™ EasYFlask™ 25 cm² flasks): DMEM (Dulbecco′s Modified Eagle′s Medium): Sourced from Corning™ (500 mL, reference: 10-017-CV), containing 4.5 g/L glucose and L-glutamine but lacking sodium pyruvate. This medium was initially used in early cultures. Sterile distilled water: Sourced from Water for Injectable Preparations Meinsol® (10 mL ampoules), used as a minimalistic medium to evaluate potential adaptability or growth under more stringent conditions. A key hypothesis of this study, shared with the genomic analysis, was that the intrinsic composition of the meteorites could act as a natural culture medium, providing the necessary environmental and chemical conditions for the development of the NSE and the detection of associated ssDNA sequences. This minimalistic approach aimed to emphasize the role of the meteorite's inherent chemical and structural features while minimizing external influences. Consistency with genomic study methodology. To ensure comparability, the culturing process, conditions, and protocols were identical to those used in the genomic study preprint (López Ramón y Cajal, 2025). This consistency highlights the strong interrelation between the microscopic characterization and genomic analysis of NSE, ensuring reliable and reproducible results across both studies. The meteorite samples used in this study were acquired from Litos, a specialized supplier of meteorites, through their official website https://www.litos.net . This supplier is known for providing verified meteorites from diverse geographical locations and classifications. Details of each meteorite, including type, fall location, and year, were confirmed through the Meteoritical Bulletin Database, where applicable. Meteorites were selected based on their qualitative compositional richness, prioritizing those classified as ordinary chondrites and carbonaceous chondrites. These types of meteorites are known for their diverse mineral and organic content, providing an optimal framework for exploring potential biological or prebiotic signatures (Sephton, 2002). These meteorites represent a diverse range of compositions and origins, providing a broad spectrum for analysis in the study. Culturing procedure. Cultures were performed at room temperature, maintained between 22 and 23°C. Meteorite fragments were placed into flasks containing the culture medium, and the flasks were vigorously shaken to facilitate the fracturing of the meteorite fragments and the release of potential internal contents. This shaking process was repeated daily at 07:00 hours to ensure consistency. Rationale for culturing mainly using sterile distilled water. A key hypothesis of this study is that the intrinsic composition of the meteorites themselves may act as a natural culture medium, providing the necessary environmental and chemical conditions for the development of the NSE and the detection of ssDNA sequences. By fracturing the meteorites, the internal material is exposed, releasing potential biologically relevant elements into the sterile distilled water medium. This minimalistic approach aims to allow the endogenous properties of the meteorites to drive the emergence and proliferation of the NSE, minimizing external influence and emphasizing the role of the meteorite's inherent chemical and structural features. Additionally, two flasks were subjected to incubation on an Eppendorf ThermoMixer F2.0, set to 56°C and 150 rpm, operating in two-hour shaking cycles. This procedure was designed to stimulate sample growth, based on observations from previous experiments conducted during the descriptive study of the NSE. The methodology aimed to alter the medium's humidity conditions, create currents within the medium through shaking, and induce controlled temperature changes. These factors had been identified in earlier studies as promoting the growth of the NSE. The cultures were maintained within a clean and disinfected cabinet, shielded from light for most of the day. During the shaking periods, however, the samples were exposed to artificial white ambient LED light. This setup was carefully designed to simulate environmental conditions conducive to the growth of the NSE. Three culture batches were carried out with fragments of meteorites. In the first batch, DMEM was used as the culture medium, while in the second and third batches, distilled water was used, identified as the optimal medium for these cultures. Each batch included three of meteorites (one per distinct meteorite, as specified later). Observations from dozens of experiments during the descriptive phase of the NSE allowed us to determine that the maximum possible number of structures was produced between 48 hours and 10 days of cultivation. The optimal period was consistently between the 7th and 10th day, so DNA extraction was scheduled accordingly. Culturing, DNA extraction and sample to microscopic study summary. Four batches of cultures were performed, with three flasks per batch, resulting in a total of 12 cultures. Each culture underwent two DNA extractions, producing a total of 24 DNA extractions. To ensure a comprehensive sampling strategy across the meteorites, one flask was cultured for each meteorite, with the exception of specific cases. For the Aiquile meteorite, three cultures were performed, while meteorites NWA 781, NWA 803, and NWA 12455 each underwent two cultures. This multi-extraction approach maximized the recovery and subsequent analysis of potential DNA from the samples, enhancing the robustness of the study. With each sample to extraction DNA we got samples to study on LOL and stain with immunofluorescence technique (see later). We studied at least between three and five samples to basis study LOL and the same number to immunofluorescence stain. After culturing, the samples were handled under strict sterile conditions. Once the culture flasks were set up and incubated, they were closed and later opened to withdraw samples for sequencing in sterile 2cc tubes, which were immediately processed in the genetics laboratory under a laminar flow hood. Samples for MTG staining were collected using a sterile syringe and sealed with a sterile needle. Additionally, unstained slides were always examined to confirm the positive staining effect of MTG before proceeding with the analysis. Microscopy. We used a Leica™ DM2000 LED microscope equipped with HIPLAN objectives: 4x, 10x, 20x, 40x, 63x, and 100x. The system included two integrated cameras: a Leica™ ICC50 W (primarily for smooth, real-time observation) and a Leica™ DMC5400 (primarily for high-resolution static imaging). The microscope was equipped with a fluorescence module featuring the I3 filter cube, which is optimized for blue excitation light. The I3 filter specifications are as follows: excitation Range: 450–490 nm (blue light); dichromatic Mirror: 510 nm and suppression Filter: Long-pass 515 nm (LP 515). The system was connected to a PC with an Intel™ Core™ i7-10700 CPU @ 2.90 GHz, 16 GB of RAM, running Windows 10 Pro 64-bit, and equipped with an NVIDIA Quadro K2000 graphics card. The setup was connected to two monitors: an LG 27UK670 4K monitor and an HP ZR2740w monitor. For image visualization, we used LAS X software, Version 3.7.4.23463 (Copyright © 2020 Leica Microsystems CMS GmbH). Images were captured and stored using the microscope's software, while video recordings were saved using Movavi Screen Recorder™, Version 21.5.0. Each image was captured using Leica software, with individual adjustments made to contrast, brightness, and exposure based on the specific characteristics of each sample. The images were saved in TIFF format to preserve maximum quality, with resolutions ranging from 5.0 MP (2592x1944 pixels) using the Leica ICC50W camera to 20.0 MP (5472x3640 pixels) on the Leica DMC5400 camera. For live imaging, the Leica ICC50W camera was consistently used at FULL HD 1080p (1920x1080 pixels). This tailored optimization and rigorous format selection allowed us to extract maximum visual information and highlight critical details in the structure of the NSE, thereby enhancing both the quality and reproducibility of the results. A second microscope, the Leica™ DMi1, was employed to visualize culture flasks. This system was equipped with HIPLAN objectives: 5x, 10x, 20x, and 40x, along with an integrated Leica™ MC 120 HD camera. Recognition of NSE developmental and adaptive phases. Based on findings of NSE in the amniotic membrane (Fig. 1 ) and human blood (Figs. 2 and 3 ) (Supplementary Video 1) (manuscripts in preparation), the NSE was identified in the following developmental and environmental adaptation phases (NSE Nomenclature) (Table 1 ): 1. Free Form (FF). Motile vesicular or slightly elongated structures, individual NSE structures. No birefringence (-). Functions as a dispersal phase, transitioning into biofilm or resistant states. 2. Biofilm Form (BF). Structured, aggregated state forming a protective matrix. Low birefringence (-) in early stages. Can evolve into a mineralized biofilm (MBF). 3. Mineralized Biofilm Form (MBF) with high birefringence (+++), indicating increased stability and resistance. 4. Resistant Form (RF) (progressive increase in structural organization and birefringence). a. Stage I. Bundled aggregate form (RF-I). Resembles simple fibers or micro-aggregated bundles arranged in linear fascicles. Variable birefringence (-/+), indicating early structural transitions. b. Stage II. Protective wall form (RF-II). Develops an external protective layer. Moderate birefringence (+), indicating molecular organization (Figs. 1 , 2 and 3 ). Can transition into Biofilm Form (BF) under specific stimuli. c. Stage III: Mineralized Laminated-Fiber Structure (RF-III). The most resistant form of NSE. High birefringence (+++), suggesting a well-organized, stable structure (Figs. 1 , 2 and 3 ). Exhibits two morphological variants: Fibrous form, resembling cellulose-like fibers. Laminated form, consisting of folded sheets with birefringence. The transition from RF-II to RF-III likely depends on environmental conditions, particularly humidity and external stimuli. Table 1 NSE forms and characteristics Form/Stage Morphology Birefringence Color Coding Function/Transition Free Form (FF) Motile vesicular or slightly elongated structures (-) Green Dispersal phase, precursor to biofilm or resistant forms Biofilm Form (BF) Flat, carpet-like structure with a defined boundary Low (-) Light green interior, intense green boundary Protective matrix, transition from free form or RF-II Mineralized Biofilm Form (MBF) Highly resistant of BF High (+++) Bluish-gray crystallized surface Resistant highly structured phase Bundled Aggregate Form (RF-I) Simple fibers or micro-aggregates arranged in linear fascicles Variable (-/+) Shades of green Intermediate resistance, structural transition Protective Wall Form (RF-II) Structure with an external protective layer Moderate (+) Bluish-gray crystallized wall Enhanced resistance, can transition to biofilm Mineralized Laminated-Fiber Structure (RF-III) Highly resistant, either fibrous (cellulose-like) or laminated with folds High (+++) Bluish-gray crystallized wall Most resistant, highly structured phase; transition depends on environmental conditions Immunofluorescence Staining: MitoTracker™ Green FM. After hundreds of observations in amniotic membranes and human blood samples, we identified the potential of MTG to effectively stain the NSE. Microscopic observations suggested a possible relationship between NSE and mitochondrial-like structures, prompting the use of MitoTracker™ Green FM (MTG) to investigate its staining properties. Experimental results demonstrated excellent staining properties of MTG for visualizing NSE in all its forms. Protocol for preparation and use of MTG. The lyophilized solid of MTG should be stored at -20°C in a desiccated environment and protected from light. Under these conditions, the reagent remains stable for up to 12 months. To prepare the stock solution, first, remove a vial of MTG from the freezer and allow it to equilibrate to room temperature in the dark for 10 minutes before reconstitution. Each vial contains 50 µg of lyophilized solid. Reconstitute the entire contents of the vial in 74.4 µL of high-quality dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. After adding DMSO, mix the solution thoroughly and let it stand at room temperature in the dark for 5 minutes to ensure complete dissolution. Preparation of working solutions. To prepare working solutions for staining, dilute the stock solution into normal culture media. The final working concentration for optimal results in staining NSE was determined to be 500 nM. For general staining guidelines: 100 nM: Add 1 µL of stock solution to 10 mL of culture media. 200 nM: Add 2 µL of stock solution to 10 mL of culture media. 300 nM: Add 3 µL of stock solution to 10 mL of culture media. Staining Procedure. Add the working solution (300–500 nM, with 500 nM as the optimal concentration) as a single drop onto one or two drops of the meteorite fragment culture medium (containing the NSE sample) placed on a glass slide. The drops of culture medium are carefully extracted using a sterile insulin syringe without the needle, and once deposited, they are gently mixed with the same syringe to ensure homogeneity. After mixing, two samples were introduced into the incubator, and one sample was observed directly under the microscope during the staining process to monitor the progression of the stain. For those samples introduced into the incubator, they were incubated at 37°C for 15–30 minutes in complete darkness to maintain optimal staining conditions and prevent photobleaching. During incubation, we monitored the humidity of the samples, and in case of desiccation, they were humidified with a single drop of sterile distilled water. For this purpose, a 10 cc ampoule of sterile distilled water was opened exclusively for humidification. Following incubation, the samples were observed and imaged live to preserve their structural and functional integrity. The concentration range used for MitoTracker™ Green FM was based on established staining protocols, which indicate that 500 nM is an optimal concentration for mitochondrial visualization (Molecular Probes, Revised June 25, 2008). Important Notes for Optimal Staining. Once reconstituted in DMSO, the stock solution should be stored at -20°C, protected from light, and used within two weeks. Freeze-thaw cycles should be avoided to maintain the reagent's integrity. For optimal results, the working solution should be freshly prepared on the same day it will be used for staining, as solutions older than 24 hours may yield inconsistent results. While the recommended range for staining mitochondria is 100–500 nM, the staining of the NSE was most effective at a concentration of 500 nM (Molecular Probes, Revised June 25, 2008). MTG: application, mechanism, and significance. MTG (Molecular Probes, Thermo Fisher Scientific) is a fluorescent dye widely used for the detection of active mitochondria in live cells. Its primary application is to assess mitochondrial abundance, morphology, and activity. Unlike some mitochondrial-specific dyes that rely exclusively on membrane potential, MTG accumulates in mitochondria independent of their membrane potential, making it particularly useful in conditions where mitochondrial membrane potential may fluctuate (Presley, Fuller, & Arriaga, 2003) (Gökerküçük, Tramier, & Bertolin, 2020). Mechanism of MTG staining. MTG is a lipophilic cationic dye that preferentially stains mitochondria due to their characteristic membrane properties and unique internal environment. However, its accumulation is not exclusive to mitochondria and can occur in other structures with similar physicochemical characteristics, such as lipid vesicles or membranous compartments containing specific phospholipids (Presley, Fuller, & Arriaga, 2003) (Zhang, Mileykovskaya, & Dowhan, 2005) (Chicco & Sparagna, 2007). The dye diffuses across membranes and is retained through molecular interactions, particularly with cardiolipin, a phospholipid enriched in mitochondrial membranes but also found in other structured lipid systems (Molecular Probes, Revised June 25, 2008). This specific affinity allows the dye to target active mitochondria, where the lipid composition and localized gradients facilitate retention (Johnson, Walsh, Bockus, & Chen, 1981). What MTG stains. MitoTracker™ Green FM (MTG) specifically stains mitochondria, but its accumulation depends on the presence of an intact and functional mitochondrial membrane (Pendergrass, Wolf, & Poot, 2004) (Xiao, Deng, Zhou, & Tan, 2016 ). Unlike potential-sensitive dyes such as tetramethylrhodamine methyl ester (TMRM) or JC-1, MTG does not require an active membrane potential for mitochondrial labelling (Scaduto & Grotyohann, 1999 ). This property makes MTG particularly useful for visualizing mitochondria in cells with variable or compromised mitochondrial activity. However, some studies indicate that MTG fluorescence can be influenced by mitochondrial depolarization and ROS levels, suggesting that under specific conditions, its labeling intensity may not be entirely membrane potential-independent (Xiao, Deng, Zhou, & Tan, 2016 ). Additionally, MTG has been reported to stain mitochondrial lipids, particularly cardiolipin, a key phospholipid involved in maintaining mitochondrial membrane structure (Zhang, Mileykovskaya, & Dowhan, 2005). This property enables researchers to assess both the spatial distribution and integrity of mitochondria in live or fixed samples. Why MTG Stains Mitochondria. The preferential staining of mitochondria by MTG is attributed to: Unique membrane composition: Mitochondrial membranes are enriched with cardiolipin, which has a high affinity for the dye. The interaction between the lipophilic dye and cardiolipin ensures selective staining. Selective retention: Once inside the mitochondria, MitoTracker Green binds to internal components, ensuring its retention despite changes in membrane potential. Gradient-driven accumulation: While the dye is independent of active potential, the local gradients and membrane permeability of mitochondria promote its selective uptake (Presley, Fuller, & Arriaga, 2003). Characteristics of non-mitochondrial structures stained by MTG. Although primarily mitochondrial-specific, MTG can stain other structures under specific conditions, provided they share certain physicochemical characteristics with mitochondria. In the case of Free Forms (FF), the uniform staining of both the external structure and internal condensations suggests a composition that interacts homogeneously with the dye. This behavior is consistent with an organized lipid or membranous environment capable of retaining MTG independently of mitochondrial activity. These include: Presence of lipid bilayers: Structures with membranes containing lipids such as cardiolipin may exhibit MTG staining. Cardiolipin, predominantly located in the inner mitochondrial membrane, plays a crucial role in maintaining mitochondrial function and structure. Alterations in cardiolipin content or composition have been associated with mitochondrial dysfunction in various tissues (Chicco & Sparagna, 2007). Electrochemical gradients: Localized electrochemical gradients or ionic conditions can enhance the retention of MTG. The mitochondrial membrane potential is a critical factor in the accumulation of certain dyes; however, MTG has been reported to stain mitochondria independent of membrane potential (Pendergrass, Wolf, & Poot, 2004). Compartmentalization: Compartments capable of trapping the dye through molecular interactions or diffusion barriers may mimic mitochondrial staining. MTG contains a mildly thiol-reactive chloromethyl moiety, allowing it to covalently bind to mitochondrial proteins and be retained within the mitochondria (Molecular Probes, Revised June 25, 2008). Molecular organization: The dye may accumulate in organized systems that replicate some biochemical properties of mitochondria, such as lipid vesicles or proto-membranes. The unique environment within mitochondria, including specific lipid compositions like cardiolipin, facilitates the selective accumulation of MTG (Chicco & Sparagna, 2007). Significance of staining. The ability of MTG to stain non-mitochondrial structures suggests that specific physicochemical properties, such as lipid composition or membrane-like organization, may influence its retention. The uniform staining observed in Free Forms (FF) and their internal structures raises questions about their composition and organization. While this observation highlights structural consistency, additional studies are required to determine whether these formations originate from purely physicochemical interactions or if other factors contribute to their characteristics. The homogeneity of MTG staining, both in the external structure and in the internal condensations, suggests that these formations share a chemically uniform composition. This observation is consistent with their role as structured compartments potentially involved in the stabilization and protection of genetic elements, rather than as metabolically active cells. This broader staining capability can be used to investigate mitochondrial-like properties in non-cellular systems, such as proto-membranes or early forms of cellular structures. The uniform staining of such systems indicates functional organization and may point to their capacity to maintain gradients or structural integrity similar to that of mitochondria. In experimental setups where non-biological systems exhibit MTG staining, the results suggest a level of organization and complexity that mimics prebiotic or proto-cellular structures (Johnson, Walsh, Bockus, & Chen, 1981) (Gökerküçük, Tramier, & Bertolin, 2020). Results The objective was to identify NSE forms originating from within the meteorite after shaking and fracturing the fragments inside the culture flask. Initially, the meteorite fragments were cultured under basal conditions and observed using an inverted microscope before proceeding with shaking. After confirming the absence of free material, vigorous and dry shaking was performed to strike the meteorite fragments against the walls of the culture flask. Intermittent observations of all fragment boundaries were conducted using the inverted microscope until the expulsion of material from the meteorite’s interior was observed (Fig. 5 ). Once the expulsion occurred, the material initially emerged in an organized manner, exhibiting intense Brownian motion (Supplementary Video 2). Within the ferrous aggregate structures extracted from the interior of the meteorite, we observed nanometric dark punctate entities displaying a dynamic transition over time: initially, many exhibited pronounced movement, characterized by oscillatory vibrations and abrupt displacements, but as the observation progressed, an increasing number of these particles gradually ceased their motion. Despite losing mobility, these particles remained clearly distinguishable from the surrounding ferrous material, retaining their individual visibility and contrast within the matrix (Supplementary Video 3). Over time, all observed particles reached a static state, appearing embedded within what visually resembled a structured ferrous sulfate aggregate, yet without fully merging into it. This progressive immobilization suggests a transition from a dispersed, dynamic phase to a structurally associated but still distinct state, likely governed by physicochemical interactions such as electrostatic forces, mineral phase transitions, or localized changes in solubility rather than autonomous biological activity. The contrast between the initially motile punctate features and their eventual stabilization, while maintaining their distinct appearance, highlights the need for further investigation into the mechanisms driving this transition. While some of these structures may share characteristics with the NSE described in this study, their behavior within the ferrous mineral matrix suggests that abiotic forces play a dominant role in their displacement and final stabilization. It subsequently diffused more freely, forming compact zones between the structures (Fig. 5 ). In this process, a Bundled Aggregate Form (RF-I) was generated from the material expelled from the meteorite’s interior (Fig. 5 ). This structure closely resembled the NSE described in human blood but featured more mineralized and compact protective walls. A key observation in the expelled ferrous material was the dynamic behavior of nanometric dark punctate entities, which initially exhibited rapid oscillatory motion and abrupt displacements. However, over time, a progressive reduction in their movement was observed, eventually leading to their complete immobilization while remaining distinguishable within the ferrous matrix. This transition raises important questions about the mechanisms governing their displacement and subsequent stabilization. The gradual cessation of movement suggests that these entities are subject to physicochemical interactions rather than autonomous biological activity. One plausible explanation is that their initial movement was driven by Brownian motion, influenced by interactions with surrounding fluid molecules. As the system reached equilibrium, the kinetic energy dissipated, leading to a decline in their mobility. Additionally, their integration into the ferrous material may result from electrostatic interactions or mineral adsorption, where attractive forces between the particles and the surrounding matrix progressively restricted their displacement until stabilization was achieved. Another possible mechanism is the formation of microstructured aggregates within the ferrous material, where particles become physically trapped in a denser, more structured mineral matrix over time. This would explain why they remain clearly visible and distinct from the surrounding material, despite no longer exhibiting independent movement. Furthermore, localized changes in solubility or ionic concentration may contribute to their immobilization, effectively anchoring them within the growing structure of the ferrous sulfate-like matrix. These findings suggest that the motion and subsequent stabilization of these punctate entities could be explained by abiotic processes. However, further research is needed to determine whether these dynamics result purely from physicochemical mechanisms or if additional factors may be involved. The progressive immobilization of these entities, while maintaining their structural integrity, highlights the complexity of the interactions occurring within the expelled material and underscores the need for further studies to elucidate the exact mechanisms driving their dynamic behavior. In some cases, the expelled structures diffused as streams that varied in compactness (Fig. 6 ), displaying a less intense Brownian motion rather than complete free Brownian movement. Instead, they formed a flow of particles within a surrounding medium, which appeared denser and more cohesive. Some of these streams remained stable for a period, while others eventually dispersed, as observed in previous cases (Fig. 6 ), before aggregating and developing into a Bundled Aggregate Form (RF-I) (Fig. 6 ). The aggregates formed from the expelled structures also showed arborescent growths with color changes, eventually forming densely packed and uniformly colored structures (Fig. 7 ). Some structures strongly resembled a Protective Wall Form (RF-II), but with mineralized walls characteristic of their meteoritic origin (Fig. 7 ). In addition to forming aggregates, the structures expelled from the meteorite progressively adhered to the plastic strips generated by scraping the fragments against the culture flask walls (Fig. 7 ). These adherent structures grew until they completely covered the plastic skeleton beneath them, preserving the original shape while continuing to grow on this foundation (Fig. 7 ). Figure 8 . - (1) A structure resembling a Protective Wall Form (RF-II) with a well-defined mineralized layer (red arrow). (2) An NSE in the RF-II/RF-III transition stage (red arrow), showing strong interaction with adhered structures collected from the meteorite (green arrow). (3) The same structure as in image (2), but under higher magnification, providing more detail. (4) A detailed examination of the NSE in RF-II/RF-III transition stage reveals zones of activity, with microvesicles (4a) observed within regions between the more mineralized walls. (5) A higher magnification view of the active region, highlighting the appearance of microvesicles, as clearly visible in the inset (5a). (6) A view of the NSE Protective Wall Form (RF-II) structure, showing a color similar to the original meteorite material, along with interactions with aggregations of structures expelled from the meteorite (green arrows). While in most cases the NSE structures exhibited a color very similar to the original meteorite fragment, we identified samples with an absolutely identical color (Figs. 9 ). Within these samples, growth of structures was observed that closely resembled those expelled from the meteorite after shaking and fragmenting. In these cases, there was no doubt that they originated from inside the meteorite (Figs. 9 and 10 ) and that there was growth occurring within the Mineralized Laminated-Fiber Structure (RF-II). This sample (Fig. 10 ) displayed significant activity, as evidenced by the genesis of a Bundled Aggregate Form (RF-I) (red arrow) originating from an aggregation. Results with MTG Staining. Different stages of the NSE were observed, and all structures were positively stained with MTG. This included both highly mineralized forms, such as the Protective Wall Form (RF-II) (Fig. 11 ), and newly formed structures like Bundled Aggregate Form (RF-I) (Fig. 12 ). The intensity of the staining was very pronounced, leaving no doubt about its positivity. Morphologies resembling worm-like or coral-like structures were identified, which are also comparable to forms occasionally found in human blood (Fig. 13 ). The staining allowed clear differentiation of the most active regions within the samples (Fig. 13 ). In recently formed Bundled Aggregate Forms (RF-I) (Fig. 14 ), the constituent bundles were distinctly visible. All structural forms were successfully stained with MTG, even the early stages of bundle formation (Fig. 15 ). Given the simplicity of some of these structures, we focused on identifying Free Forms (FF). Culture medium was extracted, stained with MTG, and meticulously examined across the entire slide under the microscope, analyzing both the full extension and height of the liquid between the coverslip and slide. Free and motile forms were successfully identified (Figs. 16 and 17 ). These Free Forms (FF) exhibited structured mobility, characterized by the presence of two or three internal rounded densities that dynamically reorganized within the structure. While their function remains unclear, further studies could explore whether they play a role in the retention or transport of genetic elements. This internal reorganization appeared to influence the overall displacement pattern, producing what can be described as oscillatory propulsive movement. At times, these internal densities generated brief linear displacements, whereas in other instances, they remained largely localized, displaying subtle vibrational shifts. Such behavior indicates that FF forms exhibit a self-organizing structural dynamic, potentially regulated by internal physicochemical interactions, facilitating the encapsulation and stabilization of ssDNA sequences under environmental conditions (Figs. 16 and 17 ) (Supplementary Video 4). Notably, in some observations, pairs of FF exhibited synchronized movement, displaying nearly identical velocities, trajectories, and stopping patterns. This phenomenon suggests the presence of an interaction mechanism between the FF, possibly mediated by electrostatic forces, fluid dynamics, or coordinated structural responses. The near-identical motion and synchronized halting behavior observed in these cases reinforce the notion that FF displacement is not solely dictated by external random forces but may involve intrinsic structural or physicochemical coupling between these entities. Further studies are needed to determine whether this coordination is an emergent property of their composition or an active process driven by self-organizing dynamics. The defining characteristics of the Free Forms (FF) include: Size: Typically, Free Forms (FF) measure approximately 3–4 µm in length and 1.5–1.8 µm in diameter. However, in some cases, when only a single internal density is observed and the interior is uniformly stained with MTG, the overall size can be as small as 1 µm. This variation suggests that FF may exist in different structural states, potentially reflecting different stages of formation, compaction, or functional adaptation. Internal Densities: Two or three actively moving, rounded internal densities measuring ~ 0.7 µm in diameter. Mobility: Exhibited oscillatory propulsive movement, with internal density shifts modulating its overall displacement. At times, these internal reorganizations resulted in linear shifts, whereas in other cases, the forms remained relatively stable while displaying localized adjustments. Such behavior may be linked to an internal structural mechanism facilitating the retention and protection of genetic material. Wall Properties: Some forms displayed greater elasticity, with flexible walls that facilitated enhanced mobility. Others appeared more rigid, with less flexible walls restricting motion. MTG Staining: All Free Forms (FF) were positively stained with MTG, which may indicate an affinity between their membrane composition and the dye, rather than direct evidence of metabolic activity. The uniform staining of both the Free Forms (FF) and their internal condensations with MTG suggests a chemically homogeneous structure rather than a biologically compartmentalized system. This observation supports the hypothesis that these formations may be governed by physicochemical interactions rather than metabolic activity, reinforcing their potential role as structured compartments for the stabilization and transport of genetic elements. These findings demonstrate the structural dynamism and morphological diversity of the NSE, particularly the distinctive properties of the Free Forms (FF). Their internal density reorganization and observed mobility suggest a complex interplay of physicochemical forces, which could contribute to the stabilization of genetic or molecular components. However, the absence of clear metabolic activity or replication indicators prevents definitive classification as biological entities. Future work should focus on experimentally testing their ability to interact with nucleic acids, as well as exploring their formation under controlled laboratory conditions to better understand their origin and functional properties. The movement of Free Forms (FF) exhibits a distinct oscillatory pattern that does not conform to conventional biological motility mechanisms. While potential explanations include physicochemical factors such as localized surface tension differentials, asymmetric Brownian motion, or electrostatic interactions, additional studies are needed to determine the underlying causes. Future research employing high-speed tracking, microfluidic assays, and computational modeling will be crucial for clarifying whether this motion results from intrinsic structural properties or external environmental influences. Discussion This study utilized an innovative optical microscopy technique (LOL) to characterize and dynamically visualize Novel Structured Entities (NSE) observed in meteorite-derived cultures. The analysis revealed four distinct morphologies: Free Form (FF), Bundled Aggregate Form (RF-I), Protective Wall Form (RF-II), and Biofilm Form (BF), all exhibiting unique organizational features. These formations, consistently detected across meteorite samples from different classifications (ordinary and carbonaceous chondrites), suggest a structured but still poorly understood phenomenon. The observed structural reorganization, particularly under MTG staining, underscores their dynamic nature. While these findings open new avenues for further investigation, the precise nature of these structures—whether they are abiotic self-assembled systems or biologically relevant entities—remains to be determined. Their presence in meteorite-derived cultures makes them an intriguing subject for studies on molecular stability and organization in extreme environments. The identification of novel ssDNA sequences in meteorite-derived cultures introduces an additional genomic aspect to the study of NSE structures. Metagenomic analysis indicates that some of these sequences lack homologs in existing databases (López Ramón y Cajal, 2025), suggesting they may correspond to previously uncharacterized genetic elements. Certain sequences exhibit conserved motifs, distinct secondary structures, and a high AT-content (54.14% in MT_PURE sequences), features sometimes linked to adaptation to extreme environments. While some extremophilic genetic elements, such as ssDNA viruses and plasmids (Krupovic, Dolja, & Kooni, 2019) (Labonté & Suttle, 2015) exhibit similar traits, there is currently no evidence indicating any functional or direct association between these ssDNA sequences and NSE structures. Future studies should prioritize experimental validation through in situ hybridization, isotopic labeling, and the controlled synthesis of analogous structures to explore potential interactions or patterns of co-occurrence with NSE. Alternatively, these sequences could be independent elements within the culture medium. Further research is required to determine their potential structural or functional significance, if any. Furthermore, the detection of shared sequence identities between the MT_TOTAL and MT_PURE datasets suggests that some of these sequences are highly conserved within the analyzed samples, reinforcing their structural and functional consistency across different meteorite-derived cultures. Notably, motif discovery analysis identified highly conserved repetitive elements that could serve as replication origins, regulatory regions, or structural domains. Given the absence of homologous sequences in known databases, future research should focus on in situ hybridization, isotopic labeling, and single-cell genomic techniques to determine whether these sequences are endogenous to NSE or originate from associated self-replicating genetic elements. Establishing a functional relationship between NSE and these ssDNA sequences will be crucial to understanding whether these structures serve as protective compartments for genetic material, facilitating its stabilization and potential replication under extreme conditions. The detection of structured ssDNA sequences in meteorite-derived cultures (López Ramón y Cajal, 2025) prompts further investigation into the stability and organization of nucleic acids in extreme environments. These sequences could hypothetically be related to prebiotic chemical processes or arise from abiotic self-assembly under specific physicochemical conditions. However, at this stage, their origin remains undetermined, and additional experimental validation is needed to discern between these possibilities. To explore these possibilities, future research should combine high-resolution molecular analysis, isotope labeling, and synthetic reconstruction of comparable structures to determine whether these sequences have prebiotic relevance or result from abiotic interactions. However, the association between these sequences and NSE structures remains highly speculative. Given the self-assembled nature of many complex physicochemical formations, it is plausible that NSE arise from abiotic self-organization processes, akin to mineralogical templating or vesicular compartmentalization observed in prebiotic chemistry. Future studies should focus on differentiating between self-organized abiotic structures and potential biological analogs, employing advanced imaging, isotopic tracing, and comparative analysis with known extremophilic systems to refine our understanding of these formations. Differential diagnosis of Free Forms (FF): a comprehensive analysis of possible candidates and insights from metagenomic data. The Free Forms (FF) identified in this study exhibit distinctive characteristics, including their dimensions (3–4 × 1.5–1.8 µm), non-linear oscillatory displacement, dynamic internal densities (~ 0.7 µm), and uniform positive staining with MTG. The mobility pattern of FF is markedly different from known microbial motility mechanisms. Unlike ultramicrobacteria, archaella-driven archaea, or magnetotactic bacteria—which rely on external appendages, gliding, or magnetic alignment for movement (Lauga & Powers, 2009) (Blakemore, 1975) (Jarrell & McBride, The surprisingly diverse ways that prokaryotes move., 2008) —FF display a motion that appears to originate from internal structural reorganization rather than active propulsion. This distinction suggests that their motility could be governed by physicochemical interactions, such as localized surface tension changes or electrostatic repulsion within the surrounding medium. Further high-resolution imaging and microfluidic assays will be essential to determine whether these movements result from biological processes or abiotic physicochemical mechanisms. In contrast, the FF display a unique irregularity in their motion, characterized by internal oscillations of their rounded densities, which drive abrupt transitions between stationary vibration and high-speed linear propulsion. This type of movement lacks parallels in known biological systems of similar dimensions. Magnetotactic bacteria, for instance, rely on static internal magnetosomes for navigation and produce a smooth helical trajectory under magnetic influence (Frankel, Bazylinski, Johnson, & Taylor, 1997 ) (Bazylinski & Frankel, 2004), while ultramicrobacteria, due to physical and energetic constraints associated with their diminutive size, are often non-motile or exhibit extremely limited motility (Dusenbery, 1997). Similarly, archaella-driven archaea use rotational mechanisms for propulsion but lack the dynamic internal reorganization observed in FF (Jarrell & McBride, The surprisingly diverse ways that prokaryotes move., 2008). In this study, we define this unique movement pattern as “oscillatory propulsive movement”, referring to the internally modulated shifts in density that result in periodic oscillations coupled with abrupt linear displacements. Unlike classical biological motility mechanisms such as flagellar rotation or gliding motility, oscillatory propulsive movement does not rely on external appendages or defined propulsion mechanisms. Instead, the movement appears to arise from intrinsic reorganizations within the structure, suggesting a physicochemical basis rather than an active biological motility system. Possible underlying mechanisms include localized surface tension variations, electrostatic interactions, or transient molecular rearrangements within the Free Forms (FF). Additionally, the irregular nature of oscillatory propulsive movement —where FF alternate between high-speed bursts and localized oscillations—contrasts sharply with random Brownian motion, which follows isotropic and thermodynamically driven patterns (Berg, 2018). Future studies employing high-speed video tracking, microfluidic assays, and computational modeling of fluid dynamics will be essential for characterizing the precise forces driving oscillatory propulsive movement and distinguishing it from biological motility systems. These experiments could also help determine whether the observed movement is modulated by external physicochemical gradients, such as ionic fluxes or energy differentials within the medium."* This behavior suggests the presence of a novel physicochemical mechanism for mobility and structural regulation, potentially influenced by internal density shifts, electrostatic interactions, or other non-biological forces adapted to extreme environments. The ability to shift between vibration and propulsion could represent an evolutionary strategy for resource acquisition, protection, or environmental adaptation. Furthermore, the combination of localized oscillations, high-speed propulsion, and flexible structural adaptations underscores the singularity of FF as a biological system. This dynamic internal movement may reflect a previously uncharacterized strategy for survival under harsh or resource-limited conditions, highlighting the need for further investigation using advanced imaging and molecular tools to elucidate its biological significance and underlying mechanisms. These features strongly suggest biological activity but raise questions about their classification and the need for a differential diagnosis. To address this, we compared FF with several bacterial and archaeal candidates based on their morphology, motility, and metabolic features (Table 2 ). Additionally, we integrated results from the metagenomic shotgun sequencing of the cultures, which provided insights into the microbial community composition and excluded many potential analogs. Nanoarchaea, such as Nanoarchaeum equitans, were considered as potential candidates due to their exceptionally small size (0.4–0.5 µm), which overlaps with the dimensions of the internal rounded densities observed in FF (Waters, y otros, 2003). These archaea are obligate symbionts, typically associated with Ignicoccus species, from which they derive essential metabolites (Podar, Makarova, Graham, Koonin, & Reysenbach, 2013) While Nanoarchaea exhibit extreme genome reduction and metabolic simplicity, they lack the dynamic motility and flexible wall properties observed in FF. Additionally, Nanoarchaea do not possess the actively moving internal rounded densities characteristic of FF, and their strict dependence on a host for metabolic activity makes MTG staining unlikely. These key differences suggest that Nanoarchaea are an unsuitable analog for FF. Ultramicrobacteria, including species such as Pelagibacter ubique, represent another group of small, streamlined organisms that could theoretically share some minimalistic characteristics with FF. These bacteria are well-adapted to oligotrophic environments, with cell sizes typically ranging from 0.3 to 0.5 µm (Giovannoni, y otros, 2005). However, ultramicrobacteria are predominantly non-motile or, at most, exhibit limited flagellar motility. Importantly, they lack visible internal structures comparable to the actively moving rounded densities observed in FF (Lauro, y otros, 2009 ). Moreover, ultramicrobacteria possess highly constrained metabolic capacities, making it unlikely that they would exhibit positive MTG staining. These key differences further differentiate ultramicrobacteria from FF and suggest that they are not a suitable analog. Magnetotactic bacteria, particularly species of the genus Magnetospirillum, share intriguing features with FF. These bacteria possess internal magnetosomes—membrane-bound crystals of magnetite or greigite arranged in chains—that enable alignment with magnetic fields (Barber-Zucker, Keren-Khadmy, & Zarivach, 2016 ). Magnetospirillum species are comparable in size, typically measuring 3–5 µm in length and 0.5–1 µm in width, and exhibit motility mediated by polar flagella (Zhang & Wu, 2020). However, their magnetosomes remain static and are arranged in linear chains, in contrast to the actively moving, rounded internal densities observed in FF. Furthermore, Magnetospirillum species exhibit a characteristic helical swimming pattern, driven by the rotation of their flagella, and do not display the oscillatory propulsive movement characteristic of FF (Reufer, y otros, 2014). Importantly, MTG staining is not typically associated with magnetotactic bacteria, as their energy generation and metabolic activity do not involve structures resembling mitochondria or related bioenergetic systems (Barber-Zucker, Keren-Khadmy, & Zarivach, 2016 ). The oscillatory movement observed in Free Forms (FF) differs from conventional microbial motility mechanisms involving external appendages, such as cilia, flagella, or gliding structures. While this movement appears to originate from internal structural dynamics, further research is necessary to determine whether it is governed by purely physicochemical forces or represents an alternative, yet uncharacterized, biological process. Another group considered was the Myxobacteria, a group of soil-dwelling bacteria known for their complex social behavior, biofilm formation, and swarming motility. While their ability to form organized aggregates may resemble the bundled forms (RF-I) observed in FF, Myxobacteria lack the dynamic individual motility displayed by FF (Cao, Dey, Vassallo, & Wall, 2015). Their swarming movement is collective, rather than individual, and they do not possess internal densities analogous to those seen in FF. Furthermore, the positive MTG staining observed in FF has not been reported in Myxobacteria, which further excludes them as candidates. Extremophilic archaea, such as Halobacterium salinarum, were also analyzed due to their adaptability to extreme environments, a trait relevant to the origin of FF in meteorite-derived cultures. These archaea exhibit flagellar motility and are metabolically active under extreme conditions, which could theoretically explain MTG staining (Oren, 2002). However, their rigid cell walls, lack of internal rounded densities, and flagellar motility patterns (linear or rotational) do not align with the elastic wall properties and “oscillatory propulsive movement” motion of FF. Consequently, extremophilic archaea are also unlikely to be related to FF. Finally, non-biological or prebiotic structures were evaluated as possible analogs. These include mineral inclusions or prebiotic aggregates that may form under specific chemical and physical conditions. Mineral inclusions can exhibit apparent mobility under microscopic observation due to Brownian motion or turbulence in liquid media (Barber & Scott, 2002 ) (Sephton, 2002) While these structures may superficially resemble FF, they lack key distinguishing properties, such as MTG staining, internally organized dynamics, and autonomous motility. These critical differences allow us to exclude non-biological structures as plausible explanations for the observed FF. The shotgun metagenomic analysis of the cultures revealed a taxonomically diverse microbial community, yet bacterial sequences constituted only 3% of the total DNA recovered. This relatively low bacterial presence makes it highly unlikely that widespread contamination could account for the observed Novel Biological Structures (NSE), including the Free Forms (FF). The taxonomic analysis identified Alphaproteobacteria as the most abundant group, particularly members of the Sphingomonadaceae family, with Sphingomonas paucimobilis (3%), Sphingomonas hankookensis (2%), and Sphingomonas alpina (1%) being the most prevalent species. These bacteria are known for their ability to survive in oligotrophic environments and metabolize complex organic compounds, suggesting a possible interaction with meteorite-derived materials. Additionally, other Alphaproteobacteria, such as Paracoccus and Erythrobacter (0.6%), were detected, along with members of Betaproteobacteria (Burkholderia), Firmicutes (Staphylococcus, Bacillus), and Actinomycetales (Streptomyces). Minor contributions from Acidobacteriota, Myxococcota, and the Terrabacteria group were also present, many of which are associated with resilience in extreme environments. Despite this microbial diversity, none of these taxa exhibit the combination of motility, self-organization, and structural characteristics observed in FF, further supporting the notion that the NSE are not attributable to known bacterial contaminants but represent an independent phenomenon within the cultures.For instance, Alphaproteobacteria and Betaproteobacteria typically lack the oscillatory propulsive movement seen in FF and instead rely on classical flagellar motility or passive dispersal (Newton, y otros, 2010) (Coenye & Vandamme, 2003). Firmicutes, such as Bacillus, are known for their ability to form spores, but their motility is flagella-driven and lacks the internal density oscillations characteristic of FF (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000). Actinomycetales, while capable of forming complex colonial structures, do not display individual motility nor dynamic internal features (Chandra & Chater, 2014 ). The metagenomic results suggest that the FF and other NSE do not easily align with any of the detected bacterial taxa, making it difficult to attribute their unique characteristics to known microorganisms. Additionally, the fact that a substantial portion of the sequences remained unclassified (51%) suggests the presence of unknown or highly divergent genetic elements, aligning with the previous discovery of novel single-stranded DNA (ssDNA) sequences in the same cultures. The combined molecular and morphological evidence suggests that the NSE, including the FF, may represent a previously uncharacterized structured system rather than artifacts of known microbial contaminants. Notably, the high percentage of unclassified sequences further supports the presence of unknown genetic elements. To elucidate their precise nature, future studies should focus on targeted molecular analyses to clarify the relationship between the FF structures and the detected ssDNA sequences. While the structural characteristics and association with novel genetic elements are provocative, definitive conclusions regarding their biological or abiotic nature cannot yet be drawn. Additional experimental evidence is necessary to clarify these intriguing associations. Table 2 Comparative Summary Table Characteristic Free Forms (FF) Nanoarchaea Ultramicrobacteria Magnetotactic Bacteria Myxobacteria Extremophilic Archaea Non-Biological Size (µm) 3–4 × 1.5–1.8 0.4–0.5 0.3–0.5 3–5 × 0.5–1 2–10 1–5 Variable Mobility “oscillatory propulsive movement”, dynamic Absent Limited Helical, magnetic-field Coordinated swarming Flagellar Apparent (Brownian) Internal Densities Motile, rounded (0.7 µm) Absent Absent Static magnetosomes Absent Absent None MTG Staining Positive Negative Negative Negative Negative Possible Negative Elasticity of Wall Variable Rigid Rigid Rigid Rigid Rigid Absent Environment Meteorite-derived cultures Host-dependent Oligotrophic waters Aquatic sediments Terrestrial soils Extreme environments Meteorites, minerals One of the striking features of the Free Forms (FF) observed in this study was their extreme rarity within the cultures. In an entire microscopic field using a 63×/0.75 objective, typically five or fewer Free Forms (FF) were observed per slide, with a maximum of up to ten. This scarcity posed significant challenges for isolating and attempting to culture them, as well as for obtaining high-quality samples for electron microscopy analysis. The isolation of FF within the cultures is an important observation that argues against the possibility of contamination. In cases of contamination, microbial growth is typically abundant and widespread, producing clusters or biofilms that are readily visible under the microscope. In contrast, the FF were singular and sparsely distributed, further reinforcing the notion that they are intrinsic to the meteorite-derived cultures and not an artifact of external contamination. The data indicate that the NSE are unlikely to be the result of external contamination and may be intrinsic to the meteorite-derived cultures; however, further confirmation is required to rule out alternative explanations Another critical consideration in evaluating the nature of the Free Forms (FF) is the potential for environmental contamination. Microbial contaminants are ubiquitous in laboratory settings and can occasionally mimic unusual structures observed in culture systems. Among the most plausible candidates for contamination are bacteria like Bacillus spp., Pseudomonas spp., and Mycoplasma spp., as well as certain archaea such as halophiles or methanogens, all of which are common in diverse environments and capable of surviving under laboratory conditions. However, a detailed analysis of these potential contaminants highlights key differences that make it unlikely for any of them to explain the observed FF structures. Bacillus spp. are well-known environmental bacteria capable of forming resilient endospores (McKenney, Driks, & Eichenberger, 2013), which could superficially resemble the internal rounded densities observed in FF. Their size range, typically between 2 and 5 µm (Errington & Aart, 2020), aligns with the dimensions of the FF. However, endospores are static structures and lack the dynamic internal movement that is a defining feature of FF. Additionally, Bacillus species are motile via flagella (Mukherjee & Kearns, 2014)but exhibit linear or random motion rather than the “oscillatory propulsive movement”, highly variable movement observed in FF. Another critical point is that if Bacillus were responsible for the FF, they would likely be present in higher numbers across the cultures. Contamination by Bacillus typically results in visible clusters or widespread presence, yet FF were isolated and required meticulous scanning of the slide to locate a single structure. Moreover, the ability of Bacillus to form spores, a static survival structure, contradicts the active, motile nature of FF (Nicholson, Munakata, Horneck, Melosh, & Setlow, 2000) (Setlow, 2014). Similarly, Pseudomonas spp. are another group of ubiquitous environmental bacteria that are commonly associated with contamination in laboratory cultures. These bacteria are known for their metabolic versatility and active flagellar motility. While Pseudomonas species are smaller than FF, typically measuring 0.5 to 0.8 µm in width and 1.5 to 3.0 µm in length (Sampedro, Parales, Krell, & Hill, 2015), their high motility might seem superficially similar. However, Pseudomonas motility is driven by flagella (Bouteiller, y otros, 2021 )and is neither “oscillatory propulsive movement” nor characterized by the changes in speed and direction that define FF movement. Additionally, Pseudomonas do not exhibit any internal structures that could correspond to the rounded, motile densities of FF. More importantly, as opportunistic contaminants, Pseudomonas would proliferate rapidly in the culture media, leading to an abundance of cells that would be easily observable across the microscope slide. The isolated presence of FF, with only one structure observed per field at high magnification, is inconsistent with Pseudomonas contamination (Silby, Winstanley, Godfrey, Lev, & Jackson, 2011) (Silby, Winstanley, Godfrey, Lev, & Jackson, 2011). Mycoplasma spp., members of the Mollicutes class, represent another group of potential contaminants due to their small size and adaptability to diverse environments. These bacteria lack a cell wall, which gives them an elastic structure, potentially resembling the flexible walls of FF. Mycoplasmas are extremely small, typically 0.15–0.3 µm in diameter (Nikfarjam & Farzaneh, 2012), significantly smaller than the 3–4 µm size observed for FF (Kasai, y otros, 2013 ). They also lack visible internal structures and are non-motile, relying on gliding rather than “oscillatory propulsive movement” movement. While Mycoplasma species are notorious contaminants in cell culture systems, their rapid growth and dispersal in media would result in widespread contamination, making their presence highly abundant in any field of view. The rarity of FF is inconsistent with the contamination patterns of Mycoplasma (Razin, Yogev, & Naot, 1998) (Drexler & Uphoff, 2002). Among archaea, halophilic species such as Halobacterium spp. are another plausible candidate due to their adaptability to extreme conditions and potential contamination during culture preparation. Halophilic archaea range in size from 1 to 5 µm (Oren, 2002), overlapping with the dimensions of FF. They are metabolically active, and some species exhibit motility via archaella, which are functionally analogous to bacterial flagella. However, their motion is typically linear or rotational(Albers & Jarrell, 2015 ) rather than “oscillatory propulsive movement”, and they lack dynamic internal densities. Methanogenic archaea, such as Methanobrevibacter spp., also merit consideration. These microorganisms are slightly smaller, measuring 0.5–2 µm (Liu & Whitman, 2008), and possess elastic cell walls that could be superficially similar to FF. However, methanogens are non-motile or exhibit minimal flagellar motility (Jarrell, Ding, Nair, & Siu, 2013 ), and their metabolic properties are incompatible with the intense MTG staining observed in FF. Furthermore, like bacterial contaminants, both halophiles and methanogens would likely appear in greater abundance if they were environmental contaminants, and their isolated presence on the slide argues against this explanation (Oren, 2002) (Garcia, Patel, & Ollivier, 2000) (Valentine, 2007). Environmental contamination is typically characterized by high cell densities and widespread distribution, yet the FF were sparsely distributed and difficult to locate, with only one structure observed per field at 63× magnification. This isolated distribution pattern, coupled with the unique morphological and dynamic features of FF, strongly argues against contamination as their source. The dynamic internal densities, “oscillatory propulsive movement” motility, and positive MTG staining further differentiate FF from common environmental microorganisms. These observations collectively support the hypothesis that FF may be intrinsic to the meteorite-derived cultures and not artifacts of laboratory contamination. Nevertheless, the possibility remains that the Free Forms (FF) could represent a contamination from an unknown extremophilic microorganism. The isolated and sparse nature of the FF, while arguing against traditional environmental contaminants, could also be consistent with the behavior of a highly specialized extremophile. These organisms are often adapted to extreme environments and may not proliferate abundantly under standard laboratory conditions, leading to their low representation in cultures. Extremophiles frequently have specific metabolic and ecological requirements that are difficult to replicate in laboratory media, which can result in their underrepresentation in experimental systems and contribute to the difficulty of their detection (Pham & Kim, 2012) (Koch, 1997). Furthermore, their unique characteristics, such as “oscillatory propulsive movement” motility, dynamic internal densities, and positive MTG staining, could reflect adaptations to environmental conditions that are not typically encountered in laboratory settings. The FF observed in this study exhibit traits reminiscent of extremophilic organisms known to thrive in Earth's most inhospitable environments. Halophilic archaea, for instance, possess specialized adaptations for survival in hypersaline environments, such as unique membrane lipids and motility via archaella, but their motion is typically linear or rotational rather than the dynamic “oscillatory propulsive movement” movement observed in FF (Oren, 2002). Similarly, thermophilic and acidophilic microorganisms, while robust and resilient to environmental stress, exhibit static structural characteristics that contrast with the flexible, dynamic features of FF. The isolation and rarity of FF in the cultures mirror the ecological behavior of extremophiles, which are often found in low abundance in their natural niches due to their highly specific environmental requirements. This raises the intriguing possibility that FF could represent a novel extremophilic structure or organism that has not yet been characterized. While the nature and origin of the FF remain uncertain, their study provides an opportunity to expand our understanding of microbial diversity and the limits of structural adaptations. Further research integrating advanced genomic and proteomic analyses, as well as environmental simulations, will be essential to determine whether the FF represent an entirely novel biological structure or a previously uncharacterized extremophilic adaptation. Furthermore, the presence of Free Forms (FF) in the same meteorite-derived cultures where novel ssDNA sequences were identified in a parallel study (López Ramón y Cajal, 2025) provides an intriguing possibility that these structures could be functionally related to the genetic elements detected. The ssDNA sequences found in these cultures, which lack similarity to known genomic databases, represent a groundbreaking discovery in themselves. The dynamic behavior, positive MTG staining, and structural uniqueness of the FF suggest that they could act as a genetic transport and stabilization system, providing a structured environment that preserves ssDNA sequences and potentially contributes to their persistence under extreme conditions. This raises the hypothesis that the FF may serve as genetic transport systemmanifestation or carrier of these sequences, offering a potential explanation for their functionality within these specialized cultures. If this connection is validated, it would represent a major advancement in understanding how novel genetic elements, such as the ssDNA sequences detected, might correspond to previously unknown biological structures. This highlights the importance of integrating morphological, molecular, and genomic data to explore the nature and role of the FF. Future studies aimed at directly linking the FF to these ssDNA sequences, through genomic localization, transcriptomic analyses, or proteomic studies, will be critical to confirm this functional relationship and further characterize their biological significance. Another key aspect of the findings in this study is the presence of distinct forms of the NSE in meteorite-derived cultures and their apparent similarities to those previously observed in human blood and amniotic membranes. The presence of these structures in vastly different contexts raises questions about their ubiquity, origin, and potential biological roles. In meteorite-derived cultures, several NSE forms were identified, including the Protective Wall Form (RF-II), Bundled Aggregate Form (RF-I), and Biofilm Form (BF). These structures exhibit morphological and staining similarities to those observed in human-derived samples. For instance, the RF-II "worm-like" and "coral-like" structures observed in meteorite cultures closely resemble analogous forms found in human blood samples. Similarly, the RF-I forms, characterized by their bundled appearance and association with dynamic vesicles, parallel the aggregative structures previously seen in amniotic membranes. Despite these similarities, there are notable differences that set the meteorite-derived NSE apart. One key difference is the degree of mineralization observed in the structures derived from meteorites. In particular, the RF-II forms found in the meteorite cultures exhibit a higher degree of rigidity and apparent mineral association, which could reflect adaptation to extraterrestrial conditions or the influence of the meteorite's mineralogical composition. This contrasts with the more flexible, biologically active RF-II forms found in human-derived samples (Supplementary Video 1). Furthermore, the association of the RF-I forms in meteorite cultures with vesicles containing unknown material raises additional questions about their biological significance. While vesicular structures have been observed in the RF-I forms in amniotic membranes, the vesicles in the meteorite-derived NSE could represent a distinct adaptation, potentially related to storage or transport of molecules under harsh environmental conditions. The presence of biofilm-like formations (BF) in meteorite-derived cultures also warrants particular attention. These structures, which exhibit pseudo-tissue-like appearances and compartmentalization, have a striking resemblance to biofilms observed in human blood. However, their sparse distribution and mineral inclusions in the meteorite samples suggest a potential functional or compositional divergence. The biofilm formations in meteorite-derived cultures might represent an adaptation to microgravity or extraterrestrial conditions, facilitating survival in nutrient-poor and physically extreme environments. The parallels and distinctions between the NSE forms observed in meteorite-derived cultures and those found in human-derived samples open intriguing questions about their origin and evolution. One possibility is that the meteorite-derived NSE could represent a convergent adaptation to environmental stressors, mirroring biological strategies observed in terrestrial systems. Alternatively, these structures might share a common origin or precursor, with their divergent features arising from environmental pressures unique to their respective contexts. The mineralized nature of the meteorite-derived NSE, coupled with their unique vesicular associations, supports the notion that these structures may have evolved in response to extraterrestrial conditions, providing a novel perspective on extremophilic adaptations. Further comparative studies between NSE forms in meteorite cultures and human-derived samples will be crucial for elucidating their shared and distinct features. These investigations could include advanced imaging techniques, compositional analyses, and genomic or proteomic studies to uncover potential evolutionary, structural, or functional connections between these fascinating structures. The fact that the flasks were observed twice daily using an inverted microscope provided valuable insights into the progression of structures present in the cultures from the very beginning and those that appeared later. This detailed monitoring allowed researchers to discern which NSE forms emerged during the frequent shaking of the flasks or through the reconfiguration of structures originating from the meteorite fragments. Consequently, this systematic observation confirmed that some of the NSE-like structures found during the experiment appeared as a result of the cultivation process and were not present initially as contamination associated with the meteorite fragments. The discovery of isolated Free Forms (FF) with unique characteristics and difficult taxonomic assignment led to the idea of exploring the "no-hits" region of the metagenomic shotgun sequencing data from these cultures. This effort aimed to identify novel or original ssDNA sequences that could potentially be associated with these distinctive motile forms. ssDNA was hypothesized as a potential source of genetic material in extreme environments due to its inherent resistance to harsh conditions and its adaptability, which has been demonstrated in certain viral and microbial systems where ssDNA plays a crucial role in survival and replication under stress (Gil, y otros, 2021) (de la Higuera & Lázaro, 2022). Remarkably, this hypothesis was validated, as approximately 80% of the DNA extracted from these cultures was identified as ssDNA, a significant proportion of which represented novel genetic elements. These findings, detailed in a previously published study (López Ramón y Cajal, 2025), provided a molecular basis for understanding the FF and opened the door to further investigations into their biological nature and potential functionality. The discovery of novel ssDNA sequences in the same meteorite-derived cultures as the Free Forms (FF) and other observed forms of the NSE suggests a direct functional or structural association between these genetic elements and the biological structures identified. These ssDNA sequences, representing approximately 80% of the DNA extracted from these cultures, exhibit unique characteristics, such as AT-rich composition, conserved secondary structures, and repetitive motifs, which may indicate their role in replication, molecular regulation, or structural stabilization. The staining of all NSE forms, including the FF, Bundled Aggregate Form (RF-I), Protective Wall Form (RF-II), and Biofilm Form (BF), with MTG highlights metabolic or bioenergetic activity that could be linked to the functionality of these novel genetic elements. The association between these novel ssDNA sequences and the diverse morphologies of the NSE observed in this study raises intriguing questions about their potential roles in biological organization, replication, and adaptation. The FF, with their dynamic motility, internal rounded densities, and elastic walls, may represent the most active and motile expression of these genetic elements. Similarly, the more organized RF-I, RF-II, and BF forms could reflect stages of aggregation, adaptation, or interaction driven by these genetic components. The observation of these structures at different stages of culture development suggests that the ssDNA sequences may play a central role in driving the formation and behavior of the NSE. These findings propose a potential evolutionary and functional link between the ssDNA sequences and the NSE forms, highlighting their adaptability to the challenging conditions within the meteorite-derived cultures. The inherent stability and adaptability of ssDNA, as noted in studies of extremophilic microorganisms and viruses, suggest that these sequences could be well-suited to environments characterized by limited resources and extreme physical or chemical conditions (Cavicchioli, Siddiqui, Andrews, & Sowers, 2002 ) (Klein, 2020). This link emphasizes the importance of further investigation into the molecular and structural interplay between the ssDNA sequences and the NSE forms, as it could unveil novel strategies of survival and replication in extreme environments. To confirm this association, future studies should employ targeted genomic and proteomic approaches to localize the ssDNA within the NSE structures and identify potential proteins or other molecules mediating their interactions. Such studies could provide critical insights into the role of ssDNA in the organization and function of these biological forms, as well as their broader implications for extremophilic biology and the potential for novel life forms in extraterrestrial-like environments. The NSE observed in meteorite-derived cultures may represent structured, adaptive formations capable of encapsulating and vehiculizing single-stranded DNA (ssDNA). These structures appear to exhibit mechanisms for maintaining the integrity of ssDNA under diverse environmental conditions, potentially acting as protective molecular carriers rather than metabolically active entities. These structures, with their diverse morphologies and dynamic behaviors, could be expressions of the unique properties of ssDNA, which appears well-suited for survival and adaptation under a wide range of environmental conditions, including potentially extreme ones. The ssDNA sequences, characterized by their AT-rich composition, conserved motifs, and secondary structural stability, likely confer molecular flexibility and functional resilience, allowing these genetic elements to persist under conditions of resource scarcity or environmental stress. The structured mobility of Free Forms (FF) is best explained by physicochemical interactions within their internal architecture, rather than by known biological motility mechanisms. Their association with ssDNA sequences, previously identified in these same cultures, suggests that these formations could serve as structured compartments that facilitate the stabilization and possible retention of autoreplicative ssDNA sequences under extreme conditions. The proposed role of NSE as protective carriers of ssDNA finds parallels in known biological and synthetic systems that encapsulate and stabilize genetic material. For instance, ssDNA viruses such as Circoviridae and Parvoviridae utilize protein-based capsids to shield their genomes from enzymatic degradation and environmental stressors, ensuring their persistence and infectivity under extreme conditions (Delwart & Li, 2012). Similarly, extracellular vesicles in eukaryotic systems transport nucleic acids within lipid bilayers, protecting them from degradation in biological fluids (Mateescu, y otros, 2017). In nanomedicine, lipid nanoparticles (LNPs) have been extensively used to encapsulate mRNA and ssDNA for targeted delivery, demonstrating that structured compartments can play a crucial role in nucleic acid stabilization (Hou, Zaks, Langer, & Dong, 2021). Given the structural organization of NSE, it is plausible that they act as physicochemically stabilized compartments for ssDNA sequences, particularly under extreme environmental conditions. Their ability to transition between dynamic Free Forms (FF) and rigid Protective Wall Forms (RF-II) suggests an adaptive structural mechanism that could regulate the accessibility, transport, or preservation of genetic material. Future studies employing fluorescence in situ hybridization (FISH) or cryo-electron microscopy (Cryo-EM) could help determine the precise localization and interaction between NSE and their associated ssDNA, providing deeper insights into their structural and functional significance. The structural organization of NSE suggests a self-assembling system capable of dynamic morphological transitions, potentially enabling the stabilization and vehiculization of genetic material in response to environmental factors. The different NSE forms—including Free Forms (FF), Bundled Aggregate Forms (RF-I), Protective Wall Forms (RF-II), and Biofilm Forms (BF)—may reflect various strategies for encapsulating and safeguarding these genetic elements from environmental stressors. The more rigid and mineralized RF-II forms could act as external protective shells, ensuring stability, while the highly dynamic and motile FF may serve as active carriers for replication and dispersal. The NSE structures may also function as organized platforms facilitating replication, molecular protection, or metabolic activity, bridging molecular innovation with structural adaptation. These findings indicate that ssDNA sequences are associated with adaptable NSE structures, suggesting a potential evolutionary mechanism that may facilitate survival under extreme conditions. The co-occurrence of these ssDNA sequences with NSE in meteorite-derived cultures points to a possible role in extremophilic adaptations. While these observations raise the possibility of novel biological interactions and adaptive strategies, further research is needed to fully determine their significance in astrobiology and prebiotic molecular evolution. The observed adaptability and structural organization of the NSE suggest intriguing parallels with prebiotic molecular assemblies, particularly protocells, coacervates, and vesicle-like systems. These self-organizing structures, widely studied in the context of the origins of life, exhibit compartmentalization, selective permeability, and dynamic interactions with their surroundings—features that are reminiscent of NSE behavior (Adamala & Szostak, 2013 ) (Chen & Walde, 2010). The ability of NSE to form aggregates, transition between different morphological states, and potentially interact with genetic material aligns with prebiotic models wherein simple molecular assemblies provided a framework for primitive biochemical processes. Notably, protocell research has demonstrated that phase-separated compartments such as coacervates can accumulate, protect, and facilitate the replication of nucleic acids, creating conditions favorable for molecular evolution (Drobot, y otros, 2018 ) (Jia, y otros, 2019). The presence of structured ssDNA sequences within NSE suggests a similar function, where the compartmentalization of genetic material within adaptable biological structures might enhance molecular stability and persistence. This hypothesis aligns with previous studies on the role of membrane-free compartments in concentrating ribonucleotides and promoting early polymerization events, which could have driven the transition from chemistry to biology (Koga, Williams, Perriman, & Mann, 2011 ) (Patel, Percivalle, Ritson, Duffy, & Sutherland, 2015). Furthermore, the role of meteorites in prebiotic chemistry is well-documented, with carbonaceous chondrites containing nucleobases, amino acids, and amphiphilic compounds capable of forming vesicle-like structures under appropriate conditions (Pearce, Pudritz, Semenov, & Henning, 2017) (Ferus, y otros, 2017 ). If the NSE observed in meteorite-derived cultures represent structures capable of molecular stabilization, replication, or selective chemical interactions, they may serve as experimental models for studying protocell-like behavior in extraterrestrial contexts. Given the environmental conditions under which these forms emerged, future studies should explore their potential stability in conditions mimicking early Earth or extraterrestrial environments, such as cycles of dehydration-rehydration, radiation exposure, and extreme temperature shifts (Rajamani, y otros, 2008) (de la Escosura, 2019). By drawing a more explicit comparison between NSE and prebiotic molecular assemblies, this study contributes to the growing body of research investigating the transition from non-living to living systems. If further investigations confirm that NSE function as primitive carriers of genetic information or as structurally adaptive molecular systems, they may represent a biological–prebiotic continuum, bridging the gap between self-assembling molecular structures and the earliest forms of cellular organization. This perspective has profound implications for astrobiology, as it suggests that similar molecular systems could emerge and persist in diverse planetary environments, including Mars, Europa, and Enceladus, where conditions favor the preservation of organic material and the emergence of molecular complexity (Sutherland, 2017) (Jordan, y otros, 2019 ). The presence of nitrogen-rich soluble organic matter in pristine samples from asteroid Bennu reinforces the notion that carbonaceous asteroids could have served as reservoirs for prebiotic chemistry. Recent studies have identified amino acids, polycyclic aromatic hydrocarbons, and nitrogen-containing heterocycles—including all five canonical nucleobases—in Bennu’s organic inventory, suggesting that these bodies contained the fundamental building blocks necessary for life (Glavin, Dworkin, Alexander, JC, & al.., 2025). These findings align with our observations of microscopic structures within meteorite-derived cultures that appear to exhibit self-organization, resilience, and interaction with selective biological stains, including MTG. The ability of these structures to retain dye suggests the presence of lipid-like components or membrane-like formations, which could indicate a form of compartmentalization, a key feature in early prebiotic evolution. Given that Bennu samples contain high concentrations of ammonia and nitrogenous compounds—both essential for amino acid and nucleotide synthesis—it is plausible that similar organic reservoirs in the early Solar System contributed to the formation of protocellular structures. Furthermore, the identification of NSE in our samples, potentially interacting with DNA probes, could indicate the presence of nucleotide-like components or their precursors. If these structures are indeed capable of binding nucleic acids, their formation in meteorite-enriched media may represent an experimental analog to early Earth conditions, where similar interactions could have contributed to the emergence of primitive genetic systems. The presence of racemic amino acids in Bennu samples further supports an abiotic origin, as no clear enantiomeric excess was detected (Glavin, Dworkin, Alexander, JC, & al.., 2025), reinforcing the idea that the organic inventory of meteorites represents a prebiotic rather than biological signature. Our findings indicate that meteorite samples subjected to aqueous conditions give rise to dynamic and organized microstructures. This observation is consistent with the presence of evaporitic minerals in Bennu samples, which suggest prolonged interaction with liquid water in its parent body (McCoy, y otros, 2025). The detection of sodium-rich phosphates, carbonates, and sulfates in Bennu’s composition supports the idea that aqueous alteration may have facilitated organic molecule stabilization and catalytic processes relevant to early biochemistry. In our study, the ability of meteorite-derived samples to form ordered assemblies in aqueous media suggests that certain minerals or organics within the meteorite may act as catalysts or scaffolds, promoting molecular self-assembly. The correlation between self-organizing structures in our study and the presence of brine-derived salts in Bennu is particularly relevant. It has been suggested that phosphate-rich environments within asteroid parent bodies could have enabled phosphorylation reactions essential for nucleotide formation (McCoy, y otros, 2025). In our case, if the NSE observed in our cultures are interacting with nucleic acids or their precursors, the meteorite material may be providing a similar chemically enriched microenvironment, where prebiotic polymerization reactions could be favored. Additionally, Bennu’s brine chemistry, which shares similarities with subsurface oceans in icy bodies such as Ceres and Enceladus, reinforces the idea that transient but chemically rich environments could support molecular complexity and potentially act as crucibles for abiogenesis. These results provide compelling empirical support for the idea that prebiotic chemical evolution is not limited to controlled laboratory settings but can emerge in naturally occurring extraterrestrial environments. The simultaneous presence of nitrogen-rich organics and aqueous-altered minerals in Bennu samples (Glavin, Dworkin, Alexander, JC, & al.., 2025) (McCoy, y otros, 2025) strengthens the argument that meteorites could serve as both molecular reservoirs and reaction environments for self-organizing structures. If, in our study, meteorite-derived material can spontaneously give rise to protocell-like formations, it follows that similar processes may have occurred on early Earth or in other planetary bodies where liquid water was transiently or persistently available. When considered alongside the chemical complexity identified in Bennu, our findings suggest that carbonaceous asteroids could have played a direct role in fostering prebiotic systems, bridging the gap between chemistry and early biology. Comparison with known biological systems: potential parallels to the NSE hypothesis. If the hypothesis that the NSE are physical, functional, and protective manifestations of ssDNA sequences is correct, it would align with several known biological systems that employ similar strategies to protect, replicate, and maintain genetic material in extreme environments. These comparisons help contextualize the potential role of the NSE in the broader landscape of biological evolution and adaptation. 1. Plasmids and ssDNA viruses: genetic elements with protective mechanisms. One of the closest biological parallels to the hypothesized NSE system is the family of ssDNA viruses and rolling-circle plasmids, which have evolved highly specialized strategies to protect and propagate their genetic material. Viruses such as Circoviridae and Parvoviridae encapsulate their ssDNA genomes within highly stable protein capsids that shield them from enzymatic degradation and environmental stressors (Delwart & Li, 2012). Similarly, rolling-circle replication (RCR) is a mechanism employed by certain bacterial and archaeal plasmids, enabling rapid replication while maintaining structural integrity in challenging environments (Khan, 1997 ). This replication strategy allows for the continuous amplification of small ssDNA molecules, a feature that could be relevant if the NSE is indeed a biological system that relies on self-replicating genetic elements. The ability of these ssDNA elements to integrate into host genomes or persist as episomal entities (Desfarges & Ciuffi, 2012) suggests a potential functional link to the observed NSE forms. If the NSE structures are indeed harboring or facilitating the replication of ssDNA, they may represent a previously uncharacterized strategy for genetic persistence in extreme environments. The presence of dynamic, vesicle-like formations within the NSE structures could further support this idea, as these features resemble the proteinaceous compartments formed by certain ssDNA viruses to compartmentalize replication processes (Kazlauskas, Varsani, Koonin, & Krupovic, 2019). 2. Nanobacteria and vesicles as protective genetic compartments. The hypothesis that the NSE structures serve as protective environments for ssDNA also aligns with historical claims about nanobacteria—submicron-sized entities that were once proposed as living microorganisms but are now more commonly regarded as self-organizing mineralized vesicles capable of enclosing genetic or biochemical material (Kajander & Ciftçioglu, 1998). These vesicles have been observed in extreme conditions, including mineral deposits and biological fluids, where they appear to form protective shells that could safeguard nucleic acids from degradation (Cíftçíoglu, Miller-Hjelle, Hjelle, & Kajander, 2002). While the existence of true nanobacteria remains controversial, the fundamental principle behind their formation—self-assembled nanoscale compartments that could house genetic material—resonates with the observed morphologies of the NSE. The presence of mineral-associated forms, such as the Protective Wall Forms (RF-II), suggests that some NSE structures may engage in a similar process of encapsulation, potentially stabilizing the ssDNA within protective layers. This would allow the genetic elements to persist in harsh conditions, much like mineralized vesicles have been proposed to do in various biological and non-biological contexts (Wu, y otros, 2016). 3. Early endosymbionts and protocells: evolutionary precursors to cellular life. Perhaps the most intriguing parallel to the NSE hypothesis comes from theories on the origin of life, particularly the role of protocells in the transition from self-replicating genetic elements to fully functional biological cells. Protocells are hypothesized to have been primitive vesicular structures that encapsulated genetic material, providing a stable microenvironment for replication and biochemical reactions (Szostak, 2012). The ability of lipid vesicles or mineral membranes to spontaneously form around nucleic acids has been demonstrated in various prebiotic chemistry experiments, suggesting that such structures could have been crucial in early molecular evolution (Maurer, Deamer, Boncella, & Monnard, 2009). If the NSE forms are indeed acting as a protective and functional system for ssDNA, they may represent an example of a naturally occurring, non-cellular genetic persistence mechanism. This could have implications for understanding the fundamental requirements for genetic continuity in extreme environments, as well as for exploring alternative models of biological organization beyond traditional cellular life. The observation that the NSE structures can exist in various morphologies—ranging from dynamic Free Forms (FF) to organized biofilms (BF)—may indicate an adaptive strategy similar to that seen in protocellular evolution, where different forms emerged in response to environmental pressures. Implications and future investigations. The similarities between the NSE structures and these established biological systems suggest that the observed formations in meteorite-derived cultures could represent a novel genetic adaptation strategy, integrating protective, replicative, and survival mechanisms. If these structures indeed function as protective compartments for ssDNA, they may provide valuable insights into how genetic material can persist outside of conventional cellular frameworks. To further validate these comparisons, future research should aim to: Determine the biochemical composition of NSE protective layers to assess their similarity to viral capsids, nanovesicles, or protocellular membranes. Identify replication-associated proteins or motifs within the ssDNA sequences that may indicate functional parallels to rolling-circle replication or viral replication systems. Investigate the self-assembly properties of the NSE structures to determine if they exhibit characteristics analogous to prebiotic vesicle formation. By exploring these aspects, we may gain deeper insights into whether the NSE represents an unknown form of biological organization, an ancient survival strategy, or even a model for the potential persistence of genetic material in extraterrestrial environments. The findings of this study on NSE share intriguing parallels with the recent discovery of nematodes revived from Siberian permafrost after 46,000 years of cryptobiosis (Shatilovich, y otros, 2023 ). Both highlight the extraordinary capacity of biological systems to adapt and persist under extreme conditions. While the nematodes rely on specialized metabolic pathways, such as the synthesis of trehalose and the glyoxylate cycle, to endure desiccation and freezing, the NSE observed in meteorite-derived cultures may represent simpler systems, potentially centered around ssDNA sequences. The resistant forms of NSE, such as the Protective Wall Forms (RF-II) and Biofilm Forms (BF), could serve a function analogous to the cryptobiotic state in nematodes, providing structural protection and stability for the ssDNA under harsh conditions. On the other hand, the dynamic Free Forms (FF), with their “oscillatory propulsive movement” motility and motile internal densities, suggest active roles in replication or dispersal, possibly reflecting an adaptive strategy distinct from the dormancy observed in nematodes. Despite these functional differences, both systems underscore the possibility of long-term survival and reactivation of biological entities in extreme or extraterrestrial environments. This parallel raises fascinating questions about the evolutionary mechanisms underpinning resilience and adaptation across vastly different biological systems, offering a unique framework for exploring the limits of life on Earth and beyond. Astrobiological implications of NSE and their association with ssDNA. The discovery of NSE in meteorite-derived cultures, particularly their structural complexity and interaction with ssDNA sequences, raises questions about the potential role of self-organizing molecular systems in prebiotic chemistry. The morphological diversity of NSE, ranging from dynamic Free Forms (FF) to highly structured Protective Wall Forms (RF-II) and Biofilm Forms (BF), suggests a capacity for environmental adaptation, although the underlying mechanisms remain to be determined. Notably, the structural features of RF-II, which exhibit mineralized-like protective shells, may play a role in shielding nucleic acids or other biomolecules from environmental stressors such as desiccation or radiation, conditions that are dominant in many extraterrestrial environments, including Mars and the icy moons of Jupiter and Saturn (Cockell, 2014). While these properties resemble survival strategies observed in extremophilic microorganisms, further analysis is required to establish their functional significance in prebiotic or astrobiological contexts (Rothschild & Mancinelli, 2001). The presence of ssDNA, constituting nearly 80% of the extracted genetic material from these cultures, further highlights the need for a deeper understanding of the molecular composition and origins of NSE. Single-stranded DNA is known for its structural flexibility and ability to form stable secondary structures under stress conditions, raising the possibility that such molecules could contribute to stability or biochemical interactions within these self-organizing systems (Pal & Levy, 2019). However, the absence of close matches in genomic databases for the ssDNA sequences identified in this study underscores the importance of additional sequencing and comparative analyses before drawing conclusions about their evolutionary or environmental adaptations. The role of ssDNA in extremophilic microorganisms and viruses adapted to harsh conditions, including high radiation and resource scarcity, suggests that further investigation could reveal whether similar molecular mechanisms are at play in NSE-like systems (Pietilä, Roine, Paulin, Kalkkinen, & Bamford, 2009). Given that extremophiles on Earth employ diverse molecular strategies to withstand extreme environments, further investigation into NSE and their interaction with ssDNA may provide insights into how self-organizing systems emerge and persist under conditions relevant to early Earth or extraterrestrial environments. While these findings do not imply a direct link to extraterrestrial life, they underscore the importance of exploring meteorite-derived materials as experimental platforms for studying molecular self-assembly and stability in simulated planetary conditions. The possibility that extremophilic molecular strategies observed on Earth could reflect evolutionary pathways suitable for survival in extraterrestrial environments remains an open question in astrobiology (Chyba & Hand, 2005). Cosmic perspective: meteorites as carriers of ancient life or prebiotic molecules. The meteorite origins of the studied NSE introduce the intriguing possibility that these structures represent biological remnants or prebiotic systems preserved from extraterrestrial environments. Meteorites are well-documented carriers of organic molecules, including amino acids, nucleobases, and lipid precursors, which form the foundational building blocks of life (Pearce, Pudritz, Semenov, & Henning, 2017). Furthermore, the preservation of these molecules in meteorites, despite their exposure to cosmic radiation and the harsh interstellar medium, suggests that protective encapsulation mechanisms—such as those potentially mirrored in RF-II and BF forms—could enable the persistence of biological systems during space travel and planetary impact (Pizzarello, 2006). The ability of the NSE to "reactivate" under laboratory conditions, forming metabolically active and motile structures, raises parallels with long-term biological dormancy and cryptobiosis observed in Earth systems (Horneck, y otros, 2008). This capacity for reactivation under favorable conditions suggests a potential mechanism for persistence in fluctuating or extreme environments. While the direct relationship between these structures and extraterrestrial conditions remains to be determined, the presence of ssDNA and dynamic biological structures in meteorite-derived cultures warrants further investigation. These findings underscore the need for integrative approaches that combine molecular, structural, and astrobiological studies to assess potential biosignatures in planetary materials, complementing efforts in missions such as the Mars Sample Return program. The concept of "Live Panspermia," as introduced in the study of ssDNA sequences in meteorite-derived cultures (López Ramón y Cajal, 2025), presents a compelling framework for understanding the biological and molecular systems potentially harbored in extraterrestrial materials. This notion posits that certain genetic elements, such as the novel ssDNA sequences identified, may have originated or been preserved in meteorites, carrying the potential for reactivation under favorable environmental conditions. The morphological and functional attributes of the NSE observed in this study align closely with the foundational ideas of Live Panspermia. Specifically, the reactivation of metabolically active, motile NSE forms under laboratory conditions mirrors the hypothesized ability of genetic systems to remain dormant during interstellar transit and subsequently regain activity when exposed to suitable conditions, such as hydration and nutrient availability. This connection reinforces the idea that NSE forms, including the Free Forms (FF) and protective wall structures, may represent physical manifestations of these genetic systems, adapted for survival in extreme or extraterrestrial environments. By linking the structural and molecular findings, this study contributes to the broader understanding of how meteorite-derived systems could inform theories on the dissemination and resilience of life across planetary boundaries. Proposed functional hypothesis of NSE and ssDNA dynamics. The observations presented in this study, alongside the genomic evidence of abundant ssDNA sequences, suggest a potential model for how NSE structures adapt and respond to environmental conditions. The Free Forms (FF) may represent short ssDNA sequences in an active state, capable of dynamic motility and interaction with their surroundings. In response to environmental cues, such as changes in nutrient availability, osmotic pressure, or other external stresses, these ssDNA sequences could reorganize or fuse, leading to the development of protective walls and more complex structures, such as Bundled Aggregate Forms (RF-I) and Protective Wall Forms (RF-II). The RF-I forms, characterized by their less mineralized and more flexible structures, may facilitate environmental interaction or proliferation within the medium. By contrast, the RF-II forms, with their highly mineralized and compact walls, appear to prioritize long-term survival under harsh conditions by offering a high degree of protection. In these quiescent states, the ssDNA sequences and their associated structures likely enter a dormant phase, where their metabolic activity, if present, is minimal, and their extraction becomes challenging. This dynamic system of structural reconfiguration, oscillating between active motile states and quiescent protective forms, represents a previously uncharacterized adaptive structural mechanism for balancing environmental interaction, stabilization, and persistence. Unlike traditional microbial systems, the ability of ssDNA-driven NSE to transition seamlessly between phases may provide a novel mechanism for genetic material preservation, particularly in extreme or resource-limited environments. This hypothesis underscores the potential significance of ssDNA as not just a genetic element but a driver of structural and physicochemical adaptability, warranting further investigation using advanced imaging and molecular techniques. Conclusions The discovery and characterization of NSE in meteorite-derived cultures suggest that these structures may incorporate a biological component, exhibiting unique structural adaptations that could be relevant to extremophilic biology. We observed NSE in several morphologies, including Free Forms (FF), Bundled Aggregate Forms (RF-I), Protective Wall Forms (RF-II), and Biofilm Forms (BF), all of which showed intense MTG staining. Because MTG is typically associated with bioenergetic activity, these observations raise the possibility that these structures may perform active biological functions; however, further investigation is required to definitively distinguish them from abiotic aggregates. The dynamic motility, flexible structural adaptations, and well-organized aggregations of these forms differentiate them from known bacterial and archaeal candidates such as ultramicrobacteria, magnetotactic bacteria, Myxobacteria, and extremophilic archaea. Moreover, metagenomic shotgun sequencing of the cultures did not identify any known microorganisms that could account for the NSE, reinforcing the idea that these structures may represent a novel biological system. The absence of contamination patterns further supports the notion that the NSE are intrinsic to the meteorite-derived cultures rather than being laboratory artifacts. A notable breakthrough was the identification of novel ssDNA sequences within the approximately 80% fraction of extracted ssDNA from the meteorite cultures. Only a subset of these sequences—originating from the no-hits region of the genomic analysis—was found to be novel (López Ramón y Cajal, 2025). These novel sequences exhibit unique AT-rich compositions, conserved motifs, and distinct secondary structural formations that suggest a high degree of adaptability. Their presence in these cultures raises the hypothesis that the various NSE morphologies may represent physical, functional, and protective manifestations of these ssDNA sequences. For example, the FF, with their “oscillatory propulsive movement” and dynamic internal densities, could function as motile carriers or dispersal forms of the ssDNA, while the more structured RF-I, RF-II, and BF forms may provide protective and adaptive environments conducive to replication and persistence. The observed association between FF and ssDNA sequences implies a potential interaction between structured compartments and genetic elements capable of autoreplication. Although the current findings do not demonstrate conventional biological activity, they offer new perspectives on how genetic material might persist and propagate under extreme conditions. This proposed link draws parallels with mechanisms such as rolling-circle replication in plasmids, encapsulation in ssDNA viruses (e.g., Circoviridae, Parvoviridae), and vesicle-like structures observed in nanobacteria. If validated, this model would describe a previously uncharacterized structured genetic system in which ssDNA elements dynamically transition between free, motile forms and structured protective aggregates, facilitating stabilization and persistence in harsh environments. These findings expand our understanding of microbial diversity and structural adaptation in extraterrestrial-like settings. The association of ssDNA sequences with NSE suggests a novel strategy for genetic persistence, structural organization, and bioenergetic function with potential implications for extremophile studies and the search for extraterrestrial life. Future research should focus on direct molecular localization of ssDNA within NSE structures, as well as transcriptomic and proteomic analyses and environmental simulations to further clarify the functional and evolutionary implications of this novel system. The integration of these approaches will be essential to determine whether NSE represent a previously unrecognized extremophilic adaptation or an entirely novel biological paradigm. In summary, the identification of metabolically active and structurally distinct NSE in meteorite-derived cultures provides a unique framework for understanding potential extraterrestrial life forms or prebiotic systems. By linking the observed morphologies to novel ssDNA sequences, this research proposes new mechanisms for genetic preservation and structural adaptation under extreme conditions. Although direct evidence linking NSE to extraterrestrial origins remains to be established, the methodologies employed in this study—including advanced microscopy and genomic analysis—offer a valuable framework for investigating biosignatures in planetary materials. These findings underscore the importance of integrating molecular, structural, and functional evidence to explore the potential diversity and resilience of life beyond Earth. Finally, the NSE structures observed in this study may represent a previously uncharacterized framework that sheds light on the stability and persistence of genetic material under extreme conditions. Their dynamic behavior, morphological diversity, and association with novel ssDNA sequences highlight their potential as a model for understanding how genetic components can be stabilized, transported, and preserved in environments that challenge conventional biological frameworks. This study emphasizes the need for an open perspective in the search for life, suggesting that early forms of life may manifest as dynamic, adaptable, and structurally simple systems. In doing so, it invites further investigation and replication by multiple research groups to fully assess the significance and universality of these observations. Limitations of the study. Although our findings offer intriguing insights into the nature of NSE, several limitations warrant cautious interpretation and further investigation. First, the microscopic characterization was limited by the resolution constraints of optical microscopy, which restricted our ability to perform a more detailed ultrastructural analysis. While MTG staining provided data consistent with metabolic activity, the exact biochemical composition and functional role of these structures remain to be clarified through additional spectroscopic and molecular approaches. Moreover, although metagenomic shotgun sequencing identified a substantial number of novel ssDNA sequences, we were unable to directly localize these sequences within the NSE structures, leaving questions about their functional integration open. The challenge of culturing or isolating Free Forms (FF) in sufficient quantities further limited the scope of controlled experimental assays to evaluate their biological properties. Additionally, the extremely low density of NSE in the cultures necessitated extensive scanning and limited the material available for comprehensive analysis, highlighting the need for more advanced enrichment techniques. Finally, despite rigorous efforts to rule out contamination, the possibility that these structures may originate from an unidentified environmental extremophile cannot be entirely excluded. Given these limitations, we emphasize the importance of independent replication by multiple research groups to validate and extend these findings, which will be critical for confirming the functional and biological significance of NSE. Finally, despite rigorous efforts to exclude contamination, we cannot entirely rule out the possibility that NSE originate from an unidentified terrestrial environmental extremophile or physicochemical processes intrinsic to the culture conditions themselves. Although MTG staining produced results consistent with metabolic activity, such staining alone does not conclusively indicate active metabolism or biological function. The precise biochemical composition, functional significance, and potential biological nature of NSE remain undetermined and require further detailed molecular and spectroscopic analyses. Moreover, although metagenomic analysis revealed novel ssDNA sequences within the same cultures, direct evidence linking these sequences functionally or causally to NSE is still lacking. Additionally, our microscopic characterization was inherently limited by the resolution constraints of optical microscopy, restricting detailed ultrastructural interpretation. The scarcity and low density of NSE within cultures further constrained the scope and depth of analysis, highlighting the need for improved enrichment and isolation methods. Despite rigorous controls to minimize contamination risks, we cannot fully exclude the possibility of an unidentified environmental origin of these structures. Future studies should include independent validation through complementary analytical techniques, enrichment procedures, and rigorous replication to clarify these uncertainties. Declarations the author declares no conflict of interest. Acknowledgments: the author would like to thank Carlos Vello Costal and his team at for their invaluable assistance. References Adamala, K., & Szostak, J. (2013 ). Nonenzymatic template-directed RNA synthesis inside model protocells. Science, 342(6162), 1098-100. doi:10.1126/science.1241888. Albers, S., & Jarrell, K. (2015 ). The archaellum: how Archaea swim. Front Microbiol., 6, 23. doi: 10.3389/fmicb.2015.00023. Aswani, K. K. (2016 ). Emerging LED Technologies for Fluorescence Microscopy. Microscopy Today, 24 (4), 22 - 27. Barber, D., & Scott, E. (2002 ). Origin of supposedly biogenic magnetite in the Martian meteorite Allan Hills 84001. Proc Natl Acad Sci U S A., 99(10), 6556-61. doi:10.1073/pnas.102045799. Barber-Zucker, S., Keren-Khadmy, N., & Zarivach, R. (2016 ). From invagination to navigation: The story of magnetosome-associated proteins in magnetotactic bacteria. Protein Sci., 25(2), 338-51. doi:10.1002/pro.2827. Bazylinski, D., & Frankel, R. (2004). Magnetosome formation in prokaryotes. Nat Rev Microbiol., 2(3), 217-30. doi:10.1038/nrmicro842. Berg, H. (2018). Random Walks in Biology: New and Expanded Edition. Kindle Edition. Princeton, Nueva Jersey.: Princeton University Press (November 20, 2018). Blakemore, R. (1975). Magnetotactic bacteria. Science, 190(4212), 377-9. doi:10.1126/science.170679 Bouteiller, M., Dupont, C., Bourigault, Y., Latour, X., Barbey, C., Konto-Ghiorghi, Y., & Merieau, A. (2021 ). Pseudomonas Flagella: Generalities and Specificities. Int J Mol Sci. , 22(7), 3337. doi:10.3390/ijms22073337. Cao, P., Dey, A., Vassallo, C., & Wall, D. (2015). How Myxobacteria Cooperate. J Mol Biol., 427(23), 3709-21. doi:10.1016/j.jmb.2015.07.022. Cavicchioli, R., Siddiqui, K., Andrews, D., & Sowers, K. (2002 ). Low-temperature extremophiles and their applications. . Curr Opin Biotechnol., 13(3), 253-61. doi:10.1016/s0958-1669(02)00317-8. Chandra, G., & Chater, K. (2014 ). Developmental biology of Streptomyces from the perspective of 100 actinobacterial genome sequences. FEMS Microbiol Rev., 38(3), 345-79. doi:10.1111/1574-6976.12047. Chaudhuri, S., Lack, J., & Coates, J. (2001). Biogenic Magnetite Formation through Anaerobic Biooxidation of Fe(II). Appl Environ Microbiol, 67(6), 2844 - 2848. doi:https://doi.org/10.1128/AEM.67.6.2844-2848.2001 Chen, I., & Walde, P. (2010). From self-assembled vesicles to protocells. . Cold Spring Harb Perspect Biol., 2(7), a002170. doi:10.1101/cshperspect.a002170. Chicco, A., & Sparagna, G. (2007). Role of cardiolipin alterations in mitochondrial dysfunction and disease. 292(1), C33-44. doi:10.1152/ajpcell.00243.2006. Chyba, C., & Hand, K. (2005). Astrobiology: The Study of the Living Universe. Annual Review of Astronomy and Astrophysics, 43, 31-74. doi:https://doi.org/10.1146/annurev.astro.43.051804.102202 Cíftçíoglu, N., Miller-Hjelle, M., Hjelle, J., & Kajander, E. (2002). Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method. Antimicrob Agents Chemother. , 46(7), 2077-86. doi:10.1128/AAC.46.7.2077-2086.2002. Cockell, C. (2014). Habitable worlds with no signs of life. Philos Trans A Math Phys Eng Sci., 372(2014), 20130082. doi:10.1098/rsta.2013.0082. Coenye, T., & Vandamme, P. (2003). Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ Microbiol., 5(9), 719-29. doi:10.1046/j.1462-2920.2003.00471.x. de la Escosura, A. (2019). The Informational Substrate of Chemical Evolution: Implications for Abiogenesis. Life (Basel)., 9(3), v. doi:10.3390/life9030066. de la Higuera, I., & Lázaro, E. (2022). Viruses in astrobiology. Front Microbiol., 13, 1032918. doi:10.3389/fmicb.2022.1032918. Delwart, E., & Li, L. (2012). Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes. Virus Res., 164((1-2)), 114-21. doi:10.1016/j.virusres.2011.11.021. Desfarges, S., & Ciuffi, A. (2012). Viral Integration and Consequences on Host Gene Expression. Viruses: Essential Agents of Life., 25, 147–75. doi:10.1007/978-94-007-4899-6_7. Drexler, H., & Uphoff, C. (2002). Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39(2), 75-90. doi:10.1023/A:1022913015916. Drobot, B., Iglesias-Artola, J., Le Vay, K., Mayr, V., Kar, M., Kreysing, M., . . . Tang, T. (2018 ). Compartmentalised RNA catalysis in membrane-free coacervate protocells. Nat Commun. , 9(1), 3643. doi:10.1038/s41467-018-06072-w. Dusenbery, D. (1997). Minimum size limit for useful locomotion by free-swimming microbes. Proc Natl Acad Sci U S A. , 94(20), 10949-54. . doi:v Errington, J., & Aart, L. (2020). Microbe Profile: Bacillus subtilis: model organism for cellular development, and industrial workhorse. Microbiology (Reading). , 166(5), 425-427. doi:10.1099/mic.0.000922. Ferus, M., Pietrucci, F., Saitta, A., Knížek, A., Kubelík, P., Ivanek, O., . . . Civiš, S. (2017 ). Formation of nucleobases in a Miller-Urey reducing atmosphere. Proc Natl Acad Sci U S A., 114(17), 4306-4311. doi:10.1073/pnas.1700010114. Frankel, R., Bazylinski, D., Johnson, M., & Taylor, B. (1997 ). Magneto-aerotaxis in marine coccoid bacteria. Biophys J. , 73(2), 994-1000. doi:10.1016/S0006-3495(97)78132-3. Garcia, J., Patel, B., & Ollivier, B. (2000). Taxonomic, phylogenetic, and ecological diversity of methanogenic Archaea. Anaerobe, 6(4), 205-26. doi:10.1006/anae.2000.0345. Gil, J., Mesa, V., Estrada-Ortiz, N., Lopez-Obando, M., Gómez, A., & Plácido, J. (2021). Viruses in Extreme Environments, Current Overview, and Biotechnological Potential. Viruses, 13(1), 81. doi:10.3390/v13010081. Giovannoni, S., Tripp, H., Givan, S., Podar, M., Vergin, K., Baptista, D., . . . Mathur, E. (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science, 309(5738), 1242-5. doi:10.1126/science.1114057. Glavin, D., Dworkin, J., Alexander, C., JC, A., & al.., e. (2025). Abundant ammonia and nitrogen-rich soluble organic matter in samples from asteroid (101955) Bennu. Nat Astron. doi:https://doi.org/10.1038/s41550-024-02472-9 Gökerküçük, E., Tramier, M., & Bertolin, G. (2020). Imaging Mitochondrial Functions: from Fluorescent Dyes to Genetically-Encoded Sensors. Genes (Basel)., 11(2), 125. doi:10.3390/genes11020125. Horneck, G., Stöffler, D., Ott, S., Hornemann, U., Cockell, C., Moeller, R., . . . Artemieva, N. (2008). Microbial rock inhabitants survive hypervelocity impacts on Mars-like host planets: first phase of lithopanspermia experimentally tested. Astrobiology, 8(1), 17-44. doi:10.1089/ast.2007.0134. Hou, X., Zaks, T., Langer, R., & Dong, Y. (2021). Lipid nanoparticles for mRNA delivery. Nat Rev Mater., 6(12), 1078-1094. doi:10.1038/s41578-021-00358-0. Jarrell, K., & McBride, M. (2008). The surprisingly diverse ways that prokaryotes move. Nat Rev Microbiol., 6(6), 466-76. doi:10.1038/nrmicro1900. Jarrell, K., Ding, Y., Nair, D., & Siu, S. (2013 ). Surface appendages of archaea: structure, function, genetics and assembly. Life (Basel)., 3(1), 86-117. doi:10.3390/life3010086. Jia, T., Chandru, K., Hongo, Y., Afrin, R., Usui, T., Myojo, K., & Cleaves, H. 2. (2019). Membraneless polyester microdroplets as primordial compartments at the origins of life. Proc Natl Acad Sci U S A., 116(32), 15830-15835. doi:10.1073/pnas.1902336116. Johnson, L., Walsh, M., Bockus, B., & Chen, L. (1981). Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. . J Cell Biol., 88(3), 526-35. doi:10.1083/jcb.88.3.526. Jordan, S., Rammu, H., Zheludev, I., Hartley, A., Maréchal, A., & Lane, N. (2019 ). Promotion of protocell self-assembly from mixed amphiphiles at the origin of life. Nat Ecol Evol., 3(12), 1705-1714. doi:10.1038/s41559-019-1015-y. Kajander, E., & Ciftçioglu, N. (1998). Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci U S A., 95(14), 8274-9. doi:10.1073/pnas.95.14.8274. Kasai, T., Nakane, D., Ishida, H., Ando, H., Kiso, M., & Miyata, M. (2013 ). Role of binding in Mycoplasma mobile and Mycoplasma pneumoniae gliding analyzed through inhibition by synthesized sialylated compounds. . J Bacteriol. , 195(3), 429-35. doi:10.1128/JB.01141-12. Kazlauskas, D., Varsani, A., Koonin, E., & Krupovic, M. (2019). Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids. Nat Commun., 10(1), 3425. doi:10.1038/s41467-019-11433-0. Khan, S. (1997 ). Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev., 61(4), 442-55. doi:10.1128/mmbr.61.4.442-455.1997. Klein, H. (2020). Stressed DNA replication generates stressed DNA. Proc Natl Acad Sci U S A., 117(19), 10108-10110. doi:10.1073/pnas.2005160117. Koch, A. (1997). Microbial physiology and ecology of slow growth. Microbiol Mol Biol Rev., 61(3), 305-18. doi:10.1128/mmbr.61.3.305-318.1997. Koga, S., Williams, D., Perriman, A., & Mann, S. (2011 ). Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. . Nat Chem., 3(9), 720-4. doi:10.1038/nchem.1110. Krupovic, M., Dolja, V., & Kooni, n. E. (2019). Origin of viruses: primordial replicators recruiting capsids from hosts. Nat Rev Microbiol., 17(7), 449-458. doi:10.1038/s41579-019-0205-6. Labonté, J., & Suttle, C. (2015). Corrigendum: Metagenomic and whole-genome analysis reveals new lineages of gokushoviruses and biogeographic separation in the sea. Front Microbiol. , 6, 114. doi:10.3389/fmicb.2015.00114. Lauga, E., & Powers, T. (2009). The hydrodynamics of swimming microorganisms. Reports on Progress in Physics, 72(9), 096601. doi:https://doi.org/10.1088/0034-4885/72/9/096601 Lauro, F., McDougald, D., Thomas, T., Williams, T., Egan, S., Rice, S., . . . Cavicchioli, R. (2009 ). The genomic basis of trophic strategy in marine bacteria. Proc Natl Acad Sci U S A., 106(37), 15527-33. doi:10.1073/pnas.0903507106. Liu, Y., & Whitman, W. (2008). Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci. , 1125, 171-89. doi:10.1196/annals.1419.019. López Ramón y Cajal, C. (2025). Discovery of single-stranded DNA in meteorite-derived cultures: evidence of novel genetic elements. bioRxiv, 633545. doi:https://doi.org/10.1101/2025.01.20.633545 Mateescu, B., Kowal, E., van, B. B., Bartel, S., Bhattacharyya, S., Buzás, E., . . . Lunav. (2017). Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles., 6(1), 1286095. doi:10.1080/20013078.2017.1286095. Maurer, S., Deamer, D., Boncella, J., & Monnard, P. (2009). Chemical evolution of amphiphiles: glycerol monoacyl derivatives stabilize plausible prebiotic membranes. Astrobiology, 9(10), 979-87. doi:10.1089/ast.2009.0384. McCoy, T., Russell, S., Zega, T., Thomas-Keprta, K., Singerling, S., Brenker, F., . . . Jones, R. (2025). An evaporite sequence from ancient brine recorded in Bennu samples. . Nature, 637((8048)), 1072-1077. doi:10.1038/s41586-024-08495-6. McKenney, P., Driks, A., & Eichenberger, P. (2013). The Bacillus subtilis endospore: assembly and functions of the multilayered coat. . Nat Rev Microbiol, 11(1), 33–44. doi:10.1038/nrmicro2921. Molecular Probes, I. (Revised June 25, 2008). MitoTracker® Mitochondrion-Selective Probes. Invitrogen Corporation. Mukherjee, S., & Kearns, D. (2014). The structure and regulation of flagella in Bacillus subtilis. Annu Rev Genet., 48, 319-40. doi:10.1146/annurev-genet-120213-092406. Newton, R., Griffin, L., Bowles, K., Meile, C., Gifford, S., Givens, C., . . . Moran, M. (2010). Genome characteristics of a generalist marine bacterial lineage. ISME J., 4(6), 784-98. doi:10.1038/ismej.2009.150. Nicholson, W., Munakata, N., Horneck, G., Melosh, H., & Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev., 64(3), 548-72. doi:10.1128/MMBR.64.3.548-572.2000. Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell J., 13(4), 203-12. Oren, A. (2002). Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J Ind Microbiol Biotechnol, 28(1), 56-63. doi:10.1038/sj/jim/7000176. Pal, A., & Levy, Y. (2019). Structure, stability and specificity of the binding of ssDNA and ssRNA with proteins. PLoS Comput Biol. , 15(4), e1006768. doi:10.1371/journal.pcbi.1006768. Patel, B., Percivalle, C., Ritson, D., Duffy, C., & Sutherland, J. (2015). Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism. Nat Chem., 7(4), 301-7. doi:10.1038/nchem.2202. Pearce, B., Pudritz, R., Semenov, D., & Henning, T. (2017). Origin of the RNA world: The fate of nucleobases in warm little ponds. Proc Natl Acad Sci U S A., 114(43), 11327-11332. doi:10.1073/pnas.1710339114. Pendergrass, W., Wolf, N., & Poot, M. (2004). Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A., 61(2), 162-9. doi:10.1002/cyto.a.20033. Pham, V., & Kim, J. (2012). Cultivation of unculturable soil bacteria. Trends Biotechnol., 30(9), 475-84. doi:10.1016/j.tibtech.2012.05.007. Pietilä, M., Roine, E., Paulin, L., Kalkkinen, N., & Bamford, D. (2009). An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope. Mol Microbiol., 72(2), 307-19. doi:10.1111/j.1365-2958.2009.06642.x Pizzarello, S. (2006). The chemistry of life's origin: a carbonaceous meteorite perspective. Acc Chem Res., 39(4), 231-7. doi:10.1021/ar050049f. Podar, M., Makarova, K., Graham, D. W., Koonin, E., & Reysenbach, A. (2013). Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park. Biol Direct., 8, 9. doi:10.1186/1745-6150-8-9. Presley, A., Fuller, K., & Arriaga, E. (2003). MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. , 793(1), 141-50. doi:doi: 10.1016/s1570-0232(03)00371-4. Rajamani, S., Vlassov, A., Benner, S., Coombs, A., Olasagasti, F., & Deamer, D. (2008). Lipid-assisted synthesis of RNA-like polymers from mononucleotides. Orig Life Evol Biosph., 38(1), 57-74. doi:10.1007/s11084-007-9113-2. Razin, S., Yogev, D., & Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev., 62(4), 1094-156. doi:10.1128/MMBR.62.4.1094-1156.1998. Reufer, M. B., Schwarz-Linek, J., Martinez, V., Morozov, A., Arlt, J., Trubitsyn, D., . . . Poon, W. (2014). Switching of Swimming Modes in Magnetospirillium gryphiswaldense. Biophys J. , 106(1), 37-46. doi:10.1016/j.bpj.2013.10.038. Rothschild, L., & Mancinelli, R. (2001). Life in extreme environments. 409(6823), 1092-101. doi:10.1038/35059215. Sampedro, I., Parales, R., Krell, T., & Hill, J. (2015). Pseudomonas chemotaxis. FEMS Microbiol Rev., 39(1), 17-46. doi:10.1111/1574-6976.12081. Scaduto, R. J., & Grotyohann, L. (1999 ). Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. . Biophys J. , 76((1 Pt 1)), 469-77. doi:10.1016/S0006-3495(99)77214-0. Sephton, M. (2002). Organic compounds in carbonaceous meteorites. Nat Prod Rep, 19(3), 292-311. doi:10.1039/b103775g. Setlow, P. (2014). Spore Resistance Properties. Microbiol Spectr., 2(5), TBS-0003-2012. doi:10.1128/microbiolspec.TBS-0003-2012. Shatilovich, A., Gade, V., Pippel, M., Hoffmeyer, T., Tchesunov, A., Stevens, L., . . . Kurzchalia, T. (2023 ). A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva. PLoS Genet., 19(7), e1010798. doi:10.1371/journal.pgen.1010798. Silby, M., Winstanley, C., Godfrey, S., Lev, y. S., & Jackson, R. (2011). Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev., 35(4), 652-80. doi:10.1111/j.1574-6976.2011.00269.x. Sutherland, J. (2017). Opinion: Studies on the origin of life — the end of the beginning. Nat Rev Chem, 1, 0012 . doi:https://doi.org/10.1038/s41570-016-0012 Szostak, J. (2012). The eightfold path to non-enzymatic RNA replication. J Syst Chem, 3, 2. doi:https://doi.org/10.1186/1759-2208-3-2 Valentine, D. (2007). Adaptations to energy stress dictate the ecology and evolution of the Archaea. Nat Rev Microbiol., 5(4), 316-23. doi:10.1038/nrmicro1619. Van Cappellen, P. (2003). Biomineralization and Global Biogeochemical Cycles. Reviews in Mineralogy and Geochemistry, 54(1), 357–381. doi:doi: https://doi.org/10.2113/0540357 Waters, E., Hohn, M., Ahel, I., Graham, D., Adams, M., Barnstead, M., . . . Noordewier, M. (2003). The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc Natl Acad Sci U S A, 100(22), 12984-8. doi:10.1073/pnas.1735403100. Wu, C., Martel, J., Wong, T., Young, D., Liu, C., Lin, C., & Young, J. (2016). Formation and characteristics of biomimetic mineralo-organic particles in natural surface water. Sci Rep., 6, 28817. doi:10.1038/srep28817 Xiao, B., Deng, X., Zhou, W., & Tan, E. (2016 ). Flow Cytometry-Based Assessment of Mitophagy Using MitoTracker. Front Cell Neurosci., 10, 76. doi:10.3389/fncel.2016.00076. Zhang, M., Mileykovskaya, E., & Dowhan, W. (2005). Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J Biol Chem., 280(33), 29403-8. doi:10.1074/jbc.M504955200. Zhang, W., & Wu, L. (2020). Flagella and Swimming Behavior of Marine Magnetotactic Bacteria. Biomolecules, 10(3), 460. doi:10.3390/biom10030460. Additional Declarations The authors declare no competing interests. Supplementary Files SupplementaryVideoMicroscopicCharacterizationandDynamicVisualizationofNSEinMeteorites.docx Supplementary Videos for: “Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures”. SupplementaryVIDEO1.mp4 Free Forms of NSE in fresh human blood. SupplementaryVIDEO2.mp4 Expulsion of material from the interior of the meteorite after agitation. SupplementaryVIDEO3.mp4 Expelled ferrous material containing free-moving black particles. SupplementaryVIDEO4.mp4 Free Form stained with MTG, featuring three dense internal nodules. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6185543","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":426043331,"identity":"b37c8714-f7cc-436a-b2ae-371b7d91f04d","order_by":0,"name":"Carlos López Ramón y Cajal","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-2912-830X","institution":"University Hospital Complex of Vigo, Spain","correspondingAuthor":true,"prefix":"","firstName":"Carlos","middleName":"López Ramón y","lastName":"Cajal","suffix":""}],"badges":[],"createdAt":"2025-03-08 18:38:53","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6185543/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6185543/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78339875,"identity":"0bc6b825-c9bd-4d73-a607-7802eaf8d2a7","added_by":"auto","created_at":"2025-03-12 08:35:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":935420,"visible":true,"origin":"","legend":"\u003cp\u003eNSE in full-term amniotic membranes. (1) Mineralized Laminated-Fiber Structure (RF-III) folded onto itself, showing bundles with varying birefringence intensities (magnified view in 1a). (2) The same NSE structure exhibits strong birefringence under polarized light microscopy. (3) Protective Wall Form (RF-II) with a well-defined external layer. (4) Birefringence of the RF-II structure, as shown in image 3. (5) Protective Wall Form (RF-II) displaying a notable interaction with the epithelium of the amniotic membranes. (6) The same structure stained with MTG, highlighting strong fluorescence and significant visualization of NSE within the membrane.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/b52a837115ad11e5b2af7cbf.png"},{"id":78341467,"identity":"5a88a950-721b-4996-b243-554c8418f86a","added_by":"auto","created_at":"2025-03-12 08:43:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1235559,"visible":true,"origin":"","legend":"\u003cp\u003eNSE in human blood. (1) A very large Mineralized Laminated-Fiber Structure (RF-III) (blue arrows) folded onto itself, forming bundles with varying birefringence intensities (magnified view in 1a). The NSE appears folded due to turbulence in the bloodstream (red arrow). (2) The same NSE structure in the RF-II stage, showing higher activity levels, as indicated by the bluish coloration. The folding is attributed to bloodstream turbulence. (3) Biofilm Form (BF) with a well-defined external layer. The sample exhibits different stages of biofilm formation on its surface, indicating periods of activity (red arrows). (4) RF-II structure with variable birefringence regions, where zones of previous biofilm formation (green arrows) display lower birefringence. (4a) Areas of past activity, forming adherent biofilms, are distinguishable by their lower birefringence. (5) An NSE in RF-II/RF-III transition stage, showing a strong interaction with adhered blood cells. The structure presents co-aggregated crystallized formations from different periods of activity, as indicated by the broad spectrum of birefringence colors. (6) An NSE in RF-I stage, exhibiting high metabolic activity (red arrow). This structure shows the formation of biofilms, either adhered to the structure or freely dispersed (green arrows), along with extensive hemolysis of adhered erythrocytes (blue arrows). (7) An NSE in the RF-I stage, exhibiting intense activity (black arrows), generating vesicles of various sizes, some of which are actively motile due to internal structural dynamics. The structure also produces biofilms at its distal end (red arrows). (8) An RF-I stage NSE with high metabolic activity, stained with MTG. The fluorescence highlights strong NSE activity and distribution.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/59921ad5f1e792fd4dc748e2.png"},{"id":78339817,"identity":"f1adeddf-02a7-416a-b1df-f08b71afaaca","added_by":"auto","created_at":"2025-03-12 08:35:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1279322,"visible":true,"origin":"","legend":"\u003cp\u003eNSE in Human Blood. (1) A very large Bundled Aggregate Form (RF-I) (red arrows), showing extensive aggregation of NSE structures within the blood sample. (2) The same NSE structure in the RF-II stage, exhibiting higher activity levels, as indicated by the bluish birefringence. (3) Biofilm Form (BF) with a well-defined external layer. The sample surface presents small black structures, suggesting periods of extreme metabolic activity. (4) Mineralized Biofilm Form (MBF) (red arrows), displaying different degrees of mineralization, indicative of a progressive maturation process. (5) Protective Wall Form (RF-II) showing significant reactivation (blue arrows), leading to the formation of small vesicles within the protective wall. The red arrow highlights hemolysis phenomena, providing further evidence of the strong interaction between NSE and blood cells, particularly erythrocytes.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/2ecd44ee0f17a72f30bd00db.png"},{"id":78341434,"identity":"fca60fb0-7193-420f-9bca-25b6d01312a1","added_by":"auto","created_at":"2025-03-12 08:43:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":441958,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative meteorite samples used in the study. The image shows fragments from various meteorites employed for culturing and subsequent analysis: Meteorite AIQUILE (ordinary chondrite H5, Bolivia), Meteorite NWA 781 (ordinary chondrite LL6, Morocco), Meteorite EUCRITA (achondrite, Algeria), Meteorite NWA 803 (ordinary chondrite L6, Northwest Africa), and Meteorite CHELYABINSK (ordinary chondrite LL5, Russia). Each fragment was carefully cleaned, disinfected, and prepared under controlled conditions for the detection and characterization of the NSE. Scale bar: 1 cm. The images represent the meteorites used and were obtained from the official website https://www.litos.net with permission.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/d36cd66edf58b8d1ef359913.png"},{"id":78341748,"identity":"8cdc50f3-c15c-459d-bd39-e18b870a6ac0","added_by":"auto","created_at":"2025-03-12 08:51:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":781892,"visible":true,"origin":"","legend":"\u003cp\u003e(1) Initiation of material expulsion from the interior of the meteorite fragment (red arrow) after agitation inside the culture flask. (2) Magnification of the expulsion zone, showing the beginning of the release process. (3) Another material expulsion site from the interior of the fragment, where the diffusion of the released content into the medium (distilled water) is evident. (3a) Close-up showing how the expelled content, initially appearing organized, begins to lose its structure as it diffuses. (4) The diffusion process continues, forming a nanometric black-speckled pattern within the structure, which appears to be ferrous in nature. (4a) Detailed view of the diffusing material. (5) The expelled material reorganizes, concentrating into more compact structures (red arrows). (6) Ultimately, the material forms a Bundled Aggregate Form (RF-I) (red arrows), originating from the expelled content of the meteorite. This structure closely resembles the previously described NSE.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/de4093c8b3a8ec9713bfb201.png"},{"id":78341437,"identity":"d2483559-ce7a-4ab6-8f11-dedaf985c741","added_by":"auto","created_at":"2025-03-12 08:43:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":719120,"visible":true,"origin":"","legend":"\u003cp\u003e(1) In this case, the expulsion of material from the meteorite's interior occurs as a compact stream with notable particle movement (resembling Brownian motion), but without free diffusion into the surrounding medium. (2) Another example of particle expulsion in a stream (red arrows), where the particles exhibit movement but do not display the completely free Brownian diffusion typically observed in nanostructures externalized from the meteorite. Instead, they show a less intense Brownian motion, moving within a denser surrounding medium that forms the stream, suggesting an interaction between the expelled particles and the surrounding fluid dynamics. (3) Even with a significant volume of expelled material, the particle streams remain adhered and compact, rather than dispersing freely into the medium. (4) Occasionally, expelled particles exhibiting Brownian motion remain in intimate association with the meteorite's surface, without freely diffusing into the medium. These particles progressively aggregate into more compact structures. (5) From these compact structures, the growth of bundles closely resembling a Bundled Aggregate Form (RF-I) can be observed. (6) Some aggregations formed from the expelled structures exhibit clear arborescent mineralization patterns, with varying degrees of mineral density. These structures develop progressively, as evidenced by distinct color changes.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/d209d3f0b752c8243bbc30b3.png"},{"id":78339846,"identity":"40921a86-2dee-4457-9343-cbc22df94ee8","added_by":"auto","created_at":"2025-03-12 08:35:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":899433,"visible":true,"origin":"","legend":"\u003cp\u003e(1) Aggregation of structures with intense color changes and arborescent mineralization (red arrow). (2) Two aggregations at different stages of mineralization, illustrating the progressive development of these structures. (3) This image shows a structure resembling a Protective Wall Form (RF-II) with a well-defined mineralized layer (red arrow). (4) Another structure with a very evident resemblance to a Protective Wall Form (RF-II) (red arrow). (5) Structures originating from the meteorite's interior progressively coat the plastic strips generated by scraping the meteorite fragments against the culture flask walls. (6) The growth of structures coating the plastic strips eventually obscures the underlying skeleton, while maintaining the original shape of the plastic strip (red arrows).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/54a49afee13cc4704ecd31c2.png"},{"id":78339822,"identity":"14e84b1d-7894-409e-8a13-42ea0acf5bf5","added_by":"auto","created_at":"2025-03-12 08:35:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":867230,"visible":true,"origin":"","legend":"\u003cp\u003e(1) A structure resembling a Protective Wall Form (RF-II) with a well-defined mineralized layer (red arrow). (2) An NSE in the RF-II/RF-III transition stage (red arrow), showing strong interaction with adhered structures collected from the meteorite (green arrow). (3) The same structure as in image (2), but under higher magnification, providing more detail. (4) A detailed examination of the NSE in RF-II/RF-III transition stage reveals zones of activity, with microvesicles (4a) observed within regions between the more mineralized walls. (5) A higher magnification view of the active region, highlighting the appearance of microvesicles, as clearly visible in the inset (5a). (6) A view of the NSE Protective Wall Form (RF-II) structure, showing a color similar to the original meteorite material, along with interactions with aggregations of structures expelled from the meteorite (green arrows).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/e34beadad134598b7622cea7.png"},{"id":78339826,"identity":"25ce9058-364d-4946-973d-1da891741b41","added_by":"auto","created_at":"2025-03-12 08:35:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":457346,"visible":true,"origin":"","legend":"\u003cp\u003eNSE structures with a color identical to the meteorite fragment. (1) A rigid Mineralized Laminated-Fiber Structure (RF-III). (2) Mineralized Laminated-Fiber Structure (RF-II) displaying internal growth of meteoritic structures (2a) and exteriorized growth extending through the wall to the outside (2b), exhibiting intense meteoritic coloration. (3) A Mineralized Laminated-Fiber Structure with higher mineralization compared to image 2, but not as extreme as in image 1. (4) A magnified detail of image 3, showing the fracture of the structure, likely caused by shaking the sample to extract it from the interior of the meteorite. (5) Mineralized Laminated-Fiber Structure (RF-II), similar to image 2, but with more evident activity, as indicated by the appearance of biofilms originating from the wall (green arrow) and growths in other areas of the wall (red arrows). (6) Mineralized Laminated-Fiber Structure (RF-II) with clear internal growth of meteoritic material (red arrows) and exterior growth (green arrows).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/c8fcc12b7438072082275e9f.png"},{"id":78341438,"identity":"2d2d3e7f-ebb3-46e1-9faa-1062174bfc09","added_by":"auto","created_at":"2025-03-12 08:43:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":739752,"visible":true,"origin":"","legend":"\u003cp\u003e(1), (2), (3), (4), and (5) Mineralized Laminated-Fiber Structure (RF-II) showing clear internal growth of meteoritic material (red arrows) at various stages and forms. (6) A detailed image illustrating the growth of a Bundled Aggregate Form (RF-I) (red arrow), originating from an aggregation.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/e06323a4babdb4e588ebece5.png"},{"id":78341436,"identity":"9465c44a-9d99-4b7c-97bf-aa8ecbdf8ab1","added_by":"auto","created_at":"2025-03-12 08:43:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":512220,"visible":true,"origin":"","legend":"\u003cp\u003eThe upper images (A, B, C) show samples extracted from the culture medium, while the corresponding lower images (a, b, c) display the same samples stained with MTG. (A, a) Biofilm Form (BF) of meteoritic origin. The biofilm is organized into independent-looking structures, resembling a pseudo-tissue appearance. The biofilm is strongly stained with MTG. (B, b) Bundled Aggregate Form (RF-I), showing nanovesicles generated by activation. The structure exhibits intense staining with MTG. (C, c) Mineralized\u003cstrong\u003e \u003c/strong\u003ebiofilm, as evidenced by the strong MTG staining.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/6d36d41367cf2645bd3793d3.png"},{"id":78342884,"identity":"6ef770a1-7ee3-40ec-94de-8c7c7bcde70f","added_by":"auto","created_at":"2025-03-12 08:59:56","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":395299,"visible":true,"origin":"","legend":"\u003cp\u003eSamples extracted from the culture medium and stained with MTG. (A) Bundled Aggregate Form (RF-I) (red arrow) showing intense staining with MTG. Fluorescent visualization highlights the structure in panels (a1) and (a2). (B) Bundled Aggregate Form (RF-I) (red arrow) with biofilm growth originating from the NSE bundles (green arrow). The structure exhibits strong MTG staining, as shown in panels (b1) and (b2).\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/78b4b3e00146c9e4692e4a2b.png"},{"id":78339844,"identity":"ade82fec-a6a9-4008-8b09-8963c4b1389d","added_by":"auto","created_at":"2025-03-12 08:35:56","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":632768,"visible":true,"origin":"","legend":"\u003cp\u003eThe upper images (A, B, C) show samples extracted from the culture medium, while the corresponding lower images (a, b, c) display the same samples stained with MTG. (A, a) Protective Wall Form (RF-II) exhibiting a \"worm-like\" structure, a morphology frequently observed in human blood or amniotic membranes. The structure is strongly stained with MTG. (B, b) Bundled Aggregate Form (RF-I) with nanovesicles, indicative of biological activity. The structure exhibits intense staining with MTG. (C, c) Protective Wall Form (RF-II) displaying a \"coral-like\" structure, where MTG staining highlights the most active regions.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/1474cf5505c9221302c2997e.png"},{"id":78339825,"identity":"d9b92872-c5fc-4058-af0e-bc7b5fd51837","added_by":"auto","created_at":"2025-03-12 08:35:55","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":475963,"visible":true,"origin":"","legend":"\u003cp\u003eThe upper images (A, B, C) show samples extracted from the culture medium, while the corresponding lower images (a, b, c) display the same samples stained with MTG. (A, a) Protective Wall Form (RF-II) exhibiting structural organization, with MTG staining highlighting its components. (B, b) Bundled Aggregate Form (RF-I) displaying significant activity, as indicated by the intense MTG staining. (C, c) Another example of Bundled Aggregate Form (RF-I), where the constituent bundles of the structure are clearly visible. MTG staining reveals its highly active nature.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/01e125e5a4925bed0750044e.png"},{"id":78339873,"identity":"cda255da-7e8d-4da8-9520-5a2c2902bc98","added_by":"auto","created_at":"2025-03-12 08:35:57","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":463096,"visible":true,"origin":"","legend":"\u003cp\u003eThe upper images (A, B, C) show samples extracted from the culture medium, while the corresponding lower images (a, b, c) display the same samples stained with MTG. (A, a) A well-defined Protective Wall Form (RF-II) exhibiting a clear structural organization, with MTG staining highlighting its structural integrity. (B, b) A transitional structure (red arrow) resembling the early stages of a Bundled Aggregate Form (RF-I). The inset highlights this free-form structure, which exhibits strong MTG staining, suggesting an active state. (C, c) Biofilm Form (BF) of meteoritic origin, characterized by its compartmentalized, pseudo-tissue-like appearance. The biofilm exhibits strong MTG staining, indicating metabolic activity.\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/1e0d19c297e4a125f1f26786.png"},{"id":78339829,"identity":"3c61fdfd-5941-43b5-8f8b-2e8bb5359b3b","added_by":"auto","created_at":"2025-03-12 08:35:56","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":354936,"visible":true,"origin":"","legend":"\u003cp\u003eFree Form (FF) stained with MTG. A free and motile structure measuring 5.25 × 1.48 μm, containing three rounded internal densities with higher fluorescence intensity. Each internal density measures approximately 0.60 μm in diameter\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/2d9541f21a2553706062df0e.png"},{"id":78341746,"identity":"01a1c24f-b409-4369-889c-d024de3f3afe","added_by":"auto","created_at":"2025-03-12 08:51:57","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":293744,"visible":true,"origin":"","legend":"\u003cp\u003eFree Form (FF) structures stained with MTG. (1) Free Form (FF) with two internal nodules, one at each end. Dimensions: 5 × 1.43 µm, with internal nodules measuring 0.88 µm in diameter. (2) Free Form (FF) with three internal nodules, one at each end and a central nodule. Dimensions: 4.46 × 1.35 µm, with internal nodule diameters of 0.79 µm. (3) Free Form (FF) with two internal nodules, one at one end and one central, exhibiting high motility with dynamic shifts between the end and center. Dimensions: 3.78 × 0.95 µm, with nodule diameters of 0.71 µm. (4) Free Form (FF) with two internal nodules, one at each end. Dimensions: 4.34 × 1.53 µm, with internal nodules measuring 0.91 µm in diameter. (5) Free Form (FF) with two clear internal nodules measuring 0.96 µm in diameter, with the emergence of a third central nodule measuring 0.63 µm. Dimensions: 4.29 × 1.48 µm. (6) Free Forms (FF) that have lost motility: (a) Dimensions: 3.13 × 1.48 µm, with a well-defined nodule (1.18 µm diameter) and a more irregular nodule at the other end. (b) Dimensions: 2.93 × 1.80 µm, with a single irregular nodule at one end measuring 0.72 µm in diameter.\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/bc9bb5ca2036c128cc7b9e35.png"},{"id":78342886,"identity":"02a7a591-f854-421e-93df-4f376879afeb","added_by":"auto","created_at":"2025-03-12 09:00:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13726854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/f98e200f-f6e0-4e3b-a69a-b6eb7471c6cf.pdf"},{"id":78341433,"identity":"833e1b0c-8412-4434-a7ea-995f8ab0c7f6","added_by":"auto","created_at":"2025-03-12 08:43:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Videos for: “Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures”.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryVideoMicroscopicCharacterizationandDynamicVisualizationofNSEinMeteorites.docx","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/4a17f1948f41fb6657b2fa52.docx"},{"id":78341465,"identity":"82a4581d-0d5f-46e0-b57a-dddc0004815e","added_by":"auto","created_at":"2025-03-12 08:43:58","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":68032316,"visible":true,"origin":"","legend":"\u003cp\u003eFree Forms of NSE in fresh human blood.\u003c/p\u003e","description":"","filename":"SupplementaryVIDEO1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/dd72cd22f52c88a8d6151656.mp4"},{"id":78341463,"identity":"01e94c00-cbbf-4b67-bd55-1ef4ac5d7983","added_by":"auto","created_at":"2025-03-12 08:43:58","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":53942460,"visible":true,"origin":"","legend":"\u003cp\u003eExpulsion of material from the interior of the meteorite after agitation.\u003c/p\u003e","description":"","filename":"SupplementaryVIDEO2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/879a0b1171cb88927dccc9f0.mp4"},{"id":78339882,"identity":"a62298e8-d11c-4960-a04e-a9b0c5492d54","added_by":"auto","created_at":"2025-03-12 08:35:58","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":94845638,"visible":true,"origin":"","legend":"\u003cp\u003eExpelled ferrous material containing free-moving black particles.\u003c/p\u003e","description":"","filename":"SupplementaryVIDEO3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/196ef7f753b40c2cabf2f897.mp4"},{"id":78339879,"identity":"0d0ffe61-990b-43cf-a632-03dc503a0833","added_by":"auto","created_at":"2025-03-12 08:35:58","extension":"mp4","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":37641391,"visible":true,"origin":"","legend":"\u003cp\u003eFree Form stained with MTG, featuring three dense internal nodules.\u003c/p\u003e","description":"","filename":"SupplementaryVIDEO4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6185543/v1/37700c6b6b90c2c7c8c61d15.mp4"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eMicroscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn biomedicine, the ongoing development of innovative methodologies has significantly enhanced the study of biological samples, leading to the discovery of novel structures and a deeper understanding of biological processes. These advancements underscore the necessity for continuous refinement of observational techniques to ensure the precise characterization of biological entities in their native state, capturing their morphology, dynamics, and functional properties while minimizing observational artifacts.\u003c/p\u003e\n\u003cp\u003eIn this study, we present an innovative microscopy approach\u0026mdash;Live Optical LED (LOL)\u0026mdash;designed for real-time visualization of biological structures in conditions that closely replicate their native environment. Unlike conventional histological techniques, which often require staining and may introduce artifacts, LOL allows direct observation without chemical modifications. This method enhances the detection of dynamic structures that could otherwise remain undetected due to sample processing limitations.\u003c/p\u003e\n\u003cp\u003eThe LOL technique operates exclusively with LED illumination, which provides superior light quality, precise wavelength control, and instantaneous activation. Unlike halogen-based systems, LED illumination, when integrated with an RGB camera, enables the acquisition of high-resolution, color-accurate images without requiring additional filtering or post-processing. This configuration ensures the preservation of the structural integrity and native characteristics of the samples. Attempts to achieve similar results with alternative light sources were unsuccessful, emphasizing the critical role of LED technology in the LOL methodology. This advanced configuration, combined with an integrated RGB camera, allows for immediate analysis of biologically active samples in real time, representing a significant advancement in the study of living tissues and biological systems (Aswani, 2016 ).\u003c/p\u003e\n\u003cp\u003eUsing the LOL methodology, we examined hundreds of freshly obtained human amniotic membranes immediately after labor, mounting them on microscope slides without fixation or staining. During these observations, we identified an uncharacterized structure interacting with both the epithelial layer and deeper regions of the amniotic membrane (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Initially termed the New Biological Structure (NBS), this formation exhibited distinctive features suggestive of a biological origin and was consistently observed across samples. In addition, it showed positive staining with MitoTracker\u0026trade; Green FM (MTG), a marker typically associated with bioenergetic activity, reinforcing its potential biological significance. This discovery prompted questions regarding its prevalence in both biological and non-biological contexts.\u003c/p\u003e\n\u003cp\u003ePrevious studies initially employed the term New Biological Structure (NBS) to describe complex morphologies observed in meteorite-derived cultures (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025). However, subsequent analyses highlighted critical uncertainties regarding the strictly biological nature of these structures. To reflect a more accurate and cautious interpretation, we now propose the term Novel Structured Entities (NSE). The NSE concept deliberately emphasizes their dynamic structural organization without presupposing biological activity or origin. Indeed, NSE could represent highly organized assemblies emerging from purely physicochemical or prebiotic self-organization processes. Although their coexistence with genetic elements is intriguing, we stress clearly that, at present, no direct functional relationship or biological origin has been confirmed. Thus, their precise nature\u0026mdash;biological, prebiotic, or purely physicochemical\u0026mdash;remains open, and our study should be viewed as foundational and exploratory, inviting further experimental scrutiny and validation.\u003c/p\u003e\n\u003cp\u003ePrevious metagenomic analyses of the same meteorite-derived cultures identified ssDNA sequences that lack homologs in current databases (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025) These sequences contain conserved structural motifs, raising questions about their origin and potential significance. Although their presence in the same cultures as NSE is an interesting observation, there is currently no evidence indicating a direct interaction or functional relationship between them. Further experimental validation is necessary to assess whether this correlation is biologically meaningful or merely a coincidental occurrence within the culture conditions.\u003c/p\u003e\n\u003cp\u003eTo further investigate this hypothesis, we examined NSE across a range of sources\u0026mdash;including human blood smears (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e), sterile plant fibers, biogenic minerals (e.g., magnetite and siderite), and notably, cultures derived from meteorite fragments. The meteorite-derived cultures were also analyzed in a parallel genomic study (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025), which confirmed an association between NSE and previously uncharacterized ssDNA sequences under identical culture conditions, thereby establishing a direct link between the structural and genetic investigations of these entities.\u003c/p\u003e\n\u003cp\u003eThe observation of NSE in meteorite-derived cultures prompts questions about their structural organization and potential stability under laboratory-simulated extreme conditions. Although their coexistence with biogenic minerals such as magnetite and siderite\u0026mdash;minerals often associated with biological processes (Chaudhuri, Lack, \u0026amp; Coates, 2001) \u0026mdash;is intriguing, we emphasize that it remains unclear whether these associations are meaningful or merely coincidental. While previous studies have suggested that biogenic minerals might contribute to structural stabilization in complex environments (Van Cappellen, 2003), further research is necessary to determine if NSE are actively involved in such stabilization processes or simply occur alongside these minerals. Likewise, the correlation observed between NSE and novel ssDNA sequences within these cultures invites further exploration; however, no direct evidence currently indicates a functional or causal relationship between them. Thus, additional rigorous experimental studies are required to clarify the precise nature of these interactions.\u003c/p\u003e\n\u003cp\u003eThis study aims to provide a detailed microscopic characterization and dynamic visualization of NSE, focusing on their detection, structural organization, and potential adaptability within meteorite-derived cultures. By utilizing the same cultures analyzed in the parallel genomic study (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025), this research complements molecular findings with high-resolution imaging, forming a cohesive framework for understanding the structural and genetic properties of NSE. Ultimately, our efforts aim to provide a detailed description and better understanding of the structural characteristics and potential adaptability of NSE observed under laboratory conditions simulating extreme environments. Although the observation of these structured entities under simulated extreme or extraterrestrial-like conditions is provocative, we explicitly underline that their exact nature remains uncertain. Our data alone cannot confirm whether NSE originate from biological, prebiotic, or purely physicochemical processes. Consequently, the interpretations presented here are preliminary and intended to stimulate rigorous, follow-up experimental studies rather than to support definitive conclusions about their biological significance or extraterrestrial origin. Future research should aim explicitly at elucidating the physicochemical conditions, molecular mechanisms, and possible functional implications underlying NSE formation and persistence.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eThe descriptive study of the NBS was conducted by the author at the \u0026Aacute;lvaro Cunqueiro Hospital in Vigo, serving as the basis for genomic analysis. The research project on the NSE was approved by the Ethics Committee for Research of Pontevedra-Vigo-Orense (Registration Codes: 2019/151 and 2019/648).\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCulturing of meteorite samples\u003c/h2\u003e\n \u003cp\u003eMeteorite fragments evaluated in the descriptive study of the NSE were cultured under the same controlled conditions and methodologies as described in the genomic study preprint (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025). The cultures were performed in sterile Nunc\u0026trade; EasYFlask\u0026trade; 25 cm\u0026sup2; flasks with Nunclon\u0026trade; Delta Surface, ensuring an optimal environment for potential growth. Two distinct culture media were employed to assess the adaptability and growth potential of biological entities within the samples. These protocols were meticulously designed to maintain a sterile and controlled environment, ensuring the reliability of subsequent DNA extraction and analysis of potential biological entities associated with the meteorite samples.\u003c/p\u003e\n \u003cp\u003eNegative controls and validation procedures.\u003c/p\u003e\n \u003cp\u003eTo ensure methodological rigor and exclude possible contamination or experimental artifacts, rigorous negative controls were implemented at key experimental steps. These controls included culturing sterile media (DMEM and sterile distilled water) without meteorite samples under identical experimental conditions (temperature, shaking, and light exposure) to evaluate the spontaneous emergence of structures. Additionally, environmental controls (culture flasks briefly exposed to laboratory air without meteorites) were cultured to rule out airborne contamination. DNA extraction was performed on these negative controls using identical protocols, confirming no detectable DNA contaminants. Furthermore, independent replicates involving meteorites of diverse origins consistently yielded NSE, whereas none appeared in the negative controls, reinforcing the reliability and specificity of our findings.\u003c/p\u003e\n \u003cp\u003eAdditionally, the reproducibility of these results was confirmed through independent replicates, using meteorites of varying origins.\u003c/p\u003e\n \u003cp\u003ePreparation of samples for cultures and culture considerations.\u003c/p\u003e\n \u003cp\u003eWe cultured one or two fragments of meteorites, each measuring 5\u0026ndash;10 mm (\u0026lt;\u0026thinsp;2 cm) per culture sample, following an exhaustive cleaning and decontamination process. The procedure involved treatment with didecyldimethylammonium chloride and non-ionic surfactants for 30 minutes, followed by immersion in pure bleach (5% sodium hypochlorite) for an additional 30 minutes and thorough rinsing with sterile water. After this process, meteorite samples were cultured under controlled laboratory conditions.\u003c/p\u003e\n \u003cp\u003eMeteorites used.\u003c/p\u003e\n \u003cp\u003eA total of seven meteorites were utilized in the study, providing a diverse range of compositions and origins (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The details of each meteorite are as follows:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 1 (AIQUILE): Type: ordinary chondrite H5. Fall location: Cochabamba, Bolivia. Confirmed fall: November 20, 2016. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 2 (NWA 781): Type: ordinary chondrite LL6. Fall location: Morocco. Fall year: 2001. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 3 (Chelyabinsk): Type: ordinary chondrite LL5. Fall location: Chelyabinskaya oblast\u0026rsquo;, Russia. Confirmed fall: February 15, 2013. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 4 (NWA 803): Type: ordinary chondrite L6. Fall location: Northwest Africa\u0026mdash;Morocco. Fall year: 2001. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 5 (NWA): Type: achondrite from the eucrite group. Fall location: Northwest Africa\u0026mdash;Algeria. Fall year: 2021. Status: not listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 6 (NWA 869): Type: ordinary chondrite L4-6. Fall location: Northwest Africa\u0026mdash;Algeria. Fall year: 2000. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMeteorite 7 (Northwest Africa, NWA 12455): Type: carbonaceous chondrite CR7. Fall location: Northwest Africa. Fall year: 2018. Status: listed in the Meteoritical Bulletin Database.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eThe meteorite samples were selected based on their compositional richness, prioritizing ordinary chondrites and carbonaceous chondrites due to their diverse mineral and organic content. These classifications provided an optimal framework for exploring potential biological or prebiotic signatures, as they are known to harbor complex organic molecules and support prebiotic chemistry (Sephton, 2002).\u003c/p\u003e\n \u003cp\u003eCulturing procedure.\u003c/p\u003e\n \u003cp\u003eCultures were performed at room temperature (22\u0026ndash;23\u0026deg;C). Meteorite fragments were placed into flasks containing the culture medium, and the flasks were vigorously shaken to facilitate the fracturing of the meteorite fragments and the release of potential internal contents. This shaking process was repeated daily at 07:00 to ensure consistency. Additionally, the cultures were evaluated at least twice daily through a detailed examination of the flasks using an inverted microscope to identify any potential forms of the NSE. These systematic evaluations aimed to detect and document the emergence of NSE structures as early as possible in the culture process.\u003c/p\u003e\n \u003cp\u003eTwo distinct culture media were utilized (20 mL per sterile Nunc\u0026trade; EasYFlask\u0026trade; 25 cm\u0026sup2; flasks):\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eDMEM (Dulbecco\u0026prime;s Modified Eagle\u0026prime;s Medium): Sourced from Corning\u0026trade; (500 mL, reference: 10-017-CV), containing 4.5 g/L glucose and L-glutamine but lacking sodium pyruvate. This medium was initially used in early cultures.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eSterile distilled water: Sourced from Water for Injectable Preparations Meinsol\u0026reg; (10 mL ampoules), used as a minimalistic medium to evaluate potential adaptability or growth under more stringent conditions.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eA key hypothesis of this study, shared with the genomic analysis, was that the intrinsic composition of the meteorites could act as a natural culture medium, providing the necessary environmental and chemical conditions for the development of the NSE and the detection of associated ssDNA sequences. This minimalistic approach aimed to emphasize the role of the meteorite\u0026apos;s inherent chemical and structural features while minimizing external influences.\u003c/p\u003e\n \u003cp\u003eConsistency with genomic study methodology.\u003c/p\u003e\n \u003cp\u003eTo ensure comparability, the culturing process, conditions, and protocols were identical to those used in the genomic study preprint (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025). This consistency highlights the strong interrelation between the microscopic characterization and genomic analysis of NSE, ensuring reliable and reproducible results across both studies.\u003c/p\u003e\n \u003cp\u003eThe meteorite samples used in this study were acquired from Litos, a specialized supplier of meteorites, through their official website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.litos.net\u003c/span\u003e\u003c/span\u003e. This supplier is known for providing verified meteorites from diverse geographical locations and classifications. Details of each meteorite, including type, fall location, and year, were confirmed through the Meteoritical Bulletin Database, where applicable.\u003c/p\u003e\n \u003cp\u003eMeteorites were selected based on their qualitative compositional richness, prioritizing those classified as ordinary chondrites and carbonaceous chondrites. These types of meteorites are known for their diverse mineral and organic content, providing an optimal framework for exploring potential biological or prebiotic signatures (Sephton, 2002). These meteorites represent a diverse range of compositions and origins, providing a broad spectrum for analysis in the study.\u003c/p\u003e\n \u003cp\u003eCulturing procedure.\u003c/p\u003e\n \u003cp\u003eCultures were performed at room temperature, maintained between 22 and 23\u0026deg;C. Meteorite fragments were placed into flasks containing the culture medium, and the flasks were vigorously shaken to facilitate the fracturing of the meteorite fragments and the release of potential internal contents. This shaking process was repeated daily at 07:00 hours to ensure consistency.\u003c/p\u003e\n \u003cp\u003eRationale for culturing mainly using sterile distilled water. A key hypothesis of this study is that the intrinsic composition of the meteorites themselves may act as a natural culture medium, providing the necessary environmental and chemical conditions for the development of the NSE and the detection of ssDNA sequences. By fracturing the meteorites, the internal material is exposed, releasing potential biologically relevant elements into the sterile distilled water medium. This minimalistic approach aims to allow the endogenous properties of the meteorites to drive the emergence and proliferation of the NSE, minimizing external influence and emphasizing the role of the meteorite\u0026apos;s inherent chemical and structural features.\u003c/p\u003e\n \u003cp\u003eAdditionally, two flasks were subjected to incubation on an Eppendorf ThermoMixer F2.0, set to 56\u0026deg;C and 150 rpm, operating in two-hour shaking cycles. This procedure was designed to stimulate sample growth, based on observations from previous experiments conducted during the descriptive study of the NSE.\u003c/p\u003e\n \u003cp\u003eThe methodology aimed to alter the medium\u0026apos;s humidity conditions, create currents within the medium through shaking, and induce controlled temperature changes. These factors had been identified in earlier studies as promoting the growth of the NSE. The cultures were maintained within a clean and disinfected cabinet, shielded from light for most of the day. During the shaking periods, however, the samples were exposed to artificial white ambient LED light. This setup was carefully designed to simulate environmental conditions conducive to the growth of the NSE.\u003c/p\u003e\n \u003cp\u003eThree culture batches were carried out with fragments of meteorites. In the first batch, DMEM was used as the culture medium, while in the second and third batches, distilled water was used, identified as the optimal medium for these cultures. Each batch included three of meteorites (one per distinct meteorite, as specified later).\u003c/p\u003e\n \u003cp\u003eObservations from dozens of experiments during the descriptive phase of the NSE allowed us to determine that the maximum possible number of structures was produced between 48 hours and 10 days of cultivation. The optimal period was consistently between the 7th and 10th day, so DNA extraction was scheduled accordingly.\u003c/p\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eCulturing, DNA extraction and sample to microscopic study summary.\u003c/p\u003e\n \u003cp\u003eFour batches of cultures were performed, with three flasks per batch, resulting in a total of 12 cultures. Each culture underwent two DNA extractions, producing a total of 24 DNA extractions. To ensure a comprehensive sampling strategy across the meteorites, one flask was cultured for each meteorite, with the exception of specific cases. For the Aiquile meteorite, three cultures were performed, while meteorites NWA 781, NWA 803, and NWA 12455 each underwent two cultures. This multi-extraction approach maximized the recovery and subsequent analysis of potential DNA from the samples, enhancing the robustness of the study.\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eWith each sample to extraction DNA we got samples to study on LOL and stain with immunofluorescence technique (see later). We studied at least between three and five samples to basis study LOL and the same number to immunofluorescence stain.\u003c/p\u003e\n \u003cp\u003eAfter culturing, the samples were handled under strict sterile conditions. Once the culture flasks were set up and incubated, they were closed and later opened to withdraw samples for sequencing in sterile 2cc tubes, which were immediately processed in the genetics laboratory under a laminar flow hood. Samples for MTG staining were collected using a sterile syringe and sealed with a sterile needle. Additionally, unstained slides were always examined to confirm the positive staining effect of MTG before proceeding with the analysis.\u003c/p\u003e\n \u003cp\u003eMicroscopy.\u003c/p\u003e\n \u003cp\u003eWe used a Leica\u0026trade; DM2000 LED microscope equipped with HIPLAN objectives: 4x, 10x, 20x, 40x, 63x, and 100x. The system included two integrated cameras: a Leica\u0026trade; ICC50 W (primarily for smooth, real-time observation) and a Leica\u0026trade; DMC5400 (primarily for high-resolution static imaging). The microscope was equipped with a fluorescence module featuring the I3 filter cube, which is optimized for blue excitation light. The I3 filter specifications are as follows: excitation Range: 450\u0026ndash;490 nm (blue light); dichromatic Mirror: 510 nm and suppression Filter: Long-pass 515 nm (LP 515).\u003c/p\u003e\n \u003cp\u003eThe system was connected to a PC with an Intel\u0026trade; Core\u0026trade; i7-10700 CPU @ 2.90 GHz, 16 GB of RAM, running Windows 10 Pro 64-bit, and equipped with an NVIDIA Quadro K2000 graphics card. The setup was connected to two monitors: an LG 27UK670 4K monitor and an HP ZR2740w monitor.\u003c/p\u003e\n \u003cp\u003eFor image visualization, we used LAS X software, Version 3.7.4.23463 (Copyright \u0026copy; 2020 Leica Microsystems CMS GmbH). Images were captured and stored using the microscope\u0026apos;s software, while video recordings were saved using Movavi Screen Recorder\u0026trade;, Version 21.5.0.\u003c/p\u003e\n \u003cp\u003eEach image was captured using Leica software, with individual adjustments made to contrast, brightness, and exposure based on the specific characteristics of each sample. The images were saved in TIFF format to preserve maximum quality, with resolutions ranging from 5.0 MP (2592x1944 pixels) using the Leica ICC50W camera to 20.0 MP (5472x3640 pixels) on the Leica DMC5400 camera. For live imaging, the Leica ICC50W camera was consistently used at FULL HD 1080p (1920x1080 pixels). This tailored optimization and rigorous format selection allowed us to extract maximum visual information and highlight critical details in the structure of the NSE, thereby enhancing both the quality and reproducibility of the results.\u003c/p\u003e\n \u003cp\u003eA second microscope, the Leica\u0026trade; DMi1, was employed to visualize culture flasks. This system was equipped with HIPLAN objectives: 5x, 10x, 20x, and 40x, along with an integrated Leica\u0026trade; MC 120 HD camera.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eRecognition of NSE developmental and adaptive phases.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBased on findings of NSE in the amniotic membrane (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and human blood (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) (Supplementary Video 1) (manuscripts in preparation), the NSE was identified in the following developmental and environmental adaptation phases (NSE Nomenclature) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e):\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e1. Free Form (FF).\u003c/strong\u003e Motile vesicular or slightly elongated structures, individual NSE structures. No birefringence (-). Functions as a dispersal phase, transitioning into biofilm or resistant states.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e2. Biofilm Form (BF).\u003c/strong\u003e Structured, aggregated state forming a protective matrix. Low birefringence (-) in early stages. Can evolve into a mineralized biofilm (MBF).\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3. Mineralized Biofilm Form (MBF)\u003c/strong\u003e with high birefringence (+++), indicating increased stability and resistance.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e4. Resistant Form (RF)\u003c/strong\u003e (progressive increase in structural organization and birefringence).\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003ea. Stage I. Bundled aggregate form (RF-I).\u003c/strong\u003e Resembles simple fibers or micro-aggregated bundles arranged in linear fascicles. Variable birefringence (-/+), indicating early structural transitions.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003eb. Stage II. Protective wall form (RF-II).\u003c/strong\u003e Develops an external protective layer. Moderate birefringence (+), indicating molecular organization (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Can transition into Biofilm Form (BF) under specific stimuli.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003ec. Stage III: Mineralized Laminated-Fiber Structure (RF-III).\u003c/strong\u003e The most resistant form of NSE. High birefringence (+++), suggesting a well-organized, stable structure (Figs. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Exhibits two morphological variants:\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eFibrous form, resembling cellulose-like fibers.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eLaminated form, consisting of folded sheets with birefringence.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eThe transition from RF-II to RF-III likely depends on environmental conditions, particularly humidity and external stimuli.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eNSE forms and characteristics\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eForm/Stage\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMorphology\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBirefringence\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eColor Coding\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFunction/Transition\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFree Form (FF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMotile vesicular or slightly elongated structures\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGreen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDispersal phase, precursor to biofilm or resistant forms\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiofilm Form (BF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlat, carpet-like structure with a defined boundary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLow (-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLight green interior, intense green boundary\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProtective matrix, transition from free form or RF-II\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMineralized Biofilm Form (MBF)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHighly resistant of BF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBluish-gray crystallized surface\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant highly structured phase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBundled Aggregate Form (RF-I)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSimple fibers or micro-aggregates arranged in linear fascicles\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVariable (-/+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eShades of green\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIntermediate resistance, structural transition\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProtective Wall Form (RF-II)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eStructure with an external protective layer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eModerate (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBluish-gray crystallized wall\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEnhanced resistance, can transition to biofilm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMineralized Laminated-Fiber Structure (RF-III)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHighly resistant, either fibrous (cellulose-like) or laminated with folds\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHigh (+++)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBluish-gray crystallized wall\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMost resistant, highly structured phase; transition depends on environmental conditions\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eImmunofluorescence Staining: MitoTracker\u0026trade; Green FM.\u003c/p\u003e\n \u003cp\u003eAfter hundreds of observations in amniotic membranes and human blood samples, we identified the potential of MTG to effectively stain the NSE. Microscopic observations suggested a possible relationship between NSE and mitochondrial-like structures, prompting the use of MitoTracker\u0026trade; Green FM (MTG) to investigate its staining properties. Experimental results demonstrated excellent staining properties of MTG for visualizing NSE in all its forms.\u003c/p\u003e\n \u003cp\u003eProtocol for preparation and use of MTG.\u003c/p\u003e\n \u003cp\u003eThe lyophilized solid of MTG should be stored at -20\u0026deg;C in a desiccated environment and protected from light. Under these conditions, the reagent remains stable for up to 12 months. To prepare the stock solution, first, remove a vial of MTG from the freezer and allow it to equilibrate to room temperature in the dark for 10 minutes before reconstitution. Each vial contains 50 \u0026micro;g of lyophilized solid. Reconstitute the entire contents of the vial in 74.4 \u0026micro;L of high-quality dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution. After adding DMSO, mix the solution thoroughly and let it stand at room temperature in the dark for 5 minutes to ensure complete dissolution.\u003c/p\u003e\n \u003cp\u003ePreparation of working solutions.\u003c/p\u003e\n \u003cp\u003eTo prepare working solutions for staining, dilute the stock solution into normal culture media. The final working concentration for optimal results in staining NSE was determined to be 500 nM.\u003c/p\u003e\n \u003cp\u003eFor general staining guidelines:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e100 nM: Add 1 \u0026micro;L of stock solution to 10 mL of culture media.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e200 nM: Add 2 \u0026micro;L of stock solution to 10 mL of culture media.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e300 nM: Add 3 \u0026micro;L of stock solution to 10 mL of culture media.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eStaining Procedure.\u003c/p\u003e\n \u003cp\u003eAdd the working solution (300\u0026ndash;500 nM, with 500 nM as the optimal concentration) as a single drop onto one or two drops of the meteorite fragment culture medium (containing the NSE sample) placed on a glass slide. The drops of culture medium are carefully extracted using a sterile insulin syringe without the needle, and once deposited, they are gently mixed with the same syringe to ensure homogeneity. After mixing, two samples were introduced into the incubator, and one sample was observed directly under the microscope during the staining process to monitor the progression of the stain.\u003c/p\u003e\n \u003cp\u003eFor those samples introduced into the incubator, they were incubated at 37\u0026deg;C for 15\u0026ndash;30 minutes in complete darkness to maintain optimal staining conditions and prevent photobleaching. During incubation, we monitored the humidity of the samples, and in case of desiccation, they were humidified with a single drop of sterile distilled water. For this purpose, a 10 cc ampoule of sterile distilled water was opened exclusively for humidification. Following incubation, the samples were observed and imaged live to preserve their structural and functional integrity. The concentration range used for MitoTracker\u0026trade; Green FM was based on established staining protocols, which indicate that 500 nM is an optimal concentration for mitochondrial visualization (Molecular Probes, Revised June 25, 2008).\u003c/p\u003e\n \u003cp\u003eImportant Notes for Optimal Staining.\u003c/p\u003e\n \u003cp\u003eOnce reconstituted in DMSO, the stock solution should be stored at -20\u0026deg;C, protected from light, and used within two weeks. Freeze-thaw cycles should be avoided to maintain the reagent\u0026apos;s integrity. For optimal results, the working solution should be freshly prepared on the same day it will be used for staining, as solutions older than 24 hours may yield inconsistent results. While the recommended range for staining mitochondria is 100\u0026ndash;500 nM, the staining of the NSE was most effective at a concentration of 500 nM (Molecular Probes, Revised June 25, 2008).\u003c/p\u003e\n \u003cp\u003eMTG: application, mechanism, and significance.\u003c/p\u003e\n \u003cp\u003eMTG (Molecular Probes, Thermo Fisher Scientific) is a fluorescent dye widely used for the detection of active mitochondria in live cells. Its primary application is to assess mitochondrial abundance, morphology, and activity. Unlike some mitochondrial-specific dyes that rely exclusively on membrane potential, MTG accumulates in mitochondria independent of their membrane potential, making it particularly useful in conditions where mitochondrial membrane potential may fluctuate (Presley, Fuller, \u0026amp; Arriaga, 2003) (G\u0026ouml;kerk\u0026uuml;\u0026ccedil;\u0026uuml;k, Tramier, \u0026amp; Bertolin, 2020).\u003c/p\u003e\n \u003cp\u003eMechanism of MTG staining.\u003c/p\u003e\n \u003cp\u003eMTG is a lipophilic cationic dye that preferentially stains mitochondria due to their characteristic membrane properties and unique internal environment. However, its accumulation is not exclusive to mitochondria and can occur in other structures with similar physicochemical characteristics, such as lipid vesicles or membranous compartments containing specific phospholipids (Presley, Fuller, \u0026amp; Arriaga, 2003) (Zhang, Mileykovskaya, \u0026amp; Dowhan, 2005) (Chicco \u0026amp; Sparagna, 2007). The dye diffuses across membranes and is retained through molecular interactions, particularly with cardiolipin, a phospholipid enriched in mitochondrial membranes but also found in other structured lipid systems (Molecular Probes, Revised June 25, 2008). This specific affinity allows the dye to target active mitochondria, where the lipid composition and localized gradients facilitate retention (Johnson, Walsh, Bockus, \u0026amp; Chen, 1981).\u003c/p\u003e\n \u003cp\u003eWhat MTG stains.\u003c/p\u003e\n \u003cp\u003eMitoTracker\u0026trade; Green FM (MTG) specifically stains mitochondria, but its accumulation depends on the presence of an intact and functional mitochondrial membrane (Pendergrass, Wolf, \u0026amp; Poot, 2004) (Xiao, Deng, Zhou, \u0026amp; Tan, 2016 ). Unlike potential-sensitive dyes such as tetramethylrhodamine methyl ester (TMRM) or JC-1, MTG does not require an active membrane potential for mitochondrial labelling (Scaduto \u0026amp; Grotyohann, 1999 ). This property makes MTG particularly useful for visualizing mitochondria in cells with variable or compromised mitochondrial activity. However, some studies indicate that MTG fluorescence can be influenced by mitochondrial depolarization and ROS levels, suggesting that under specific conditions, its labeling intensity may not be entirely membrane potential-independent (Xiao, Deng, Zhou, \u0026amp; Tan, 2016 ). Additionally, MTG has been reported to stain mitochondrial lipids, particularly cardiolipin, a key phospholipid involved in maintaining mitochondrial membrane structure (Zhang, Mileykovskaya, \u0026amp; Dowhan, 2005). This property enables researchers to assess both the spatial distribution and integrity of mitochondria in live or fixed samples.\u003c/p\u003e\n \u003cp\u003eWhy MTG Stains Mitochondria. The preferential staining of mitochondria by MTG is attributed to:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eUnique membrane composition: Mitochondrial membranes are enriched with cardiolipin, which has a high affinity for the dye. The interaction between the lipophilic dye and cardiolipin ensures selective staining.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eSelective retention: Once inside the mitochondria, MitoTracker Green binds to internal components, ensuring its retention despite changes in membrane potential.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eGradient-driven accumulation: While the dye is independent of active potential, the local gradients and membrane permeability of mitochondria promote its selective uptake (Presley, Fuller, \u0026amp; Arriaga, 2003).\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eCharacteristics of non-mitochondrial structures stained by MTG.\u003c/p\u003e\n \u003cp\u003eAlthough primarily mitochondrial-specific, MTG can stain other structures under specific conditions, provided they share certain physicochemical characteristics with mitochondria. In the case of Free Forms (FF), the uniform staining of both the external structure and internal condensations suggests a composition that interacts homogeneously with the dye. This behavior is consistent with an organized lipid or membranous environment capable of retaining MTG independently of mitochondrial activity. These include:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003ePresence of lipid bilayers: Structures with membranes containing lipids such as cardiolipin may exhibit MTG staining. Cardiolipin, predominantly located in the inner mitochondrial membrane, plays a crucial role in maintaining mitochondrial function and structure. Alterations in cardiolipin content or composition have been associated with mitochondrial dysfunction in various tissues (Chicco \u0026amp; Sparagna, 2007).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eElectrochemical gradients: Localized electrochemical gradients or ionic conditions can enhance the retention of MTG. The mitochondrial membrane potential is a critical factor in the accumulation of certain dyes; however, MTG has been reported to stain mitochondria independent of membrane potential (Pendergrass, Wolf, \u0026amp; Poot, 2004).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eCompartmentalization: Compartments capable of trapping the dye through molecular interactions or diffusion barriers may mimic mitochondrial staining. MTG contains a mildly thiol-reactive chloromethyl moiety, allowing it to covalently bind to mitochondrial proteins and be retained within the mitochondria (Molecular Probes, Revised June 25, 2008).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMolecular organization: The dye may accumulate in organized systems that replicate some biochemical properties of mitochondria, such as lipid vesicles or proto-membranes. The unique environment within mitochondria, including specific lipid compositions like cardiolipin, facilitates the selective accumulation of MTG (Chicco \u0026amp; Sparagna, 2007).\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eSignificance of staining.\u003c/p\u003e\n \u003cp\u003eThe ability of MTG to stain non-mitochondrial structures suggests that specific physicochemical properties, such as lipid composition or membrane-like organization, may influence its retention. The uniform staining observed in Free Forms (FF) and their internal structures raises questions about their composition and organization. While this observation highlights structural consistency, additional studies are required to determine whether these formations originate from purely physicochemical interactions or if other factors contribute to their characteristics. The homogeneity of MTG staining, both in the external structure and in the internal condensations, suggests that these formations share a chemically uniform composition. This observation is consistent with their role as structured compartments potentially involved in the stabilization and protection of genetic elements, rather than as metabolically active cells. This broader staining capability can be used to investigate mitochondrial-like properties in non-cellular systems, such as proto-membranes or early forms of cellular structures. The uniform staining of such systems indicates functional organization and may point to their capacity to maintain gradients or structural integrity similar to that of mitochondria. In experimental setups where non-biological systems exhibit MTG staining, the results suggest a level of organization and complexity that mimics prebiotic or proto-cellular structures (Johnson, Walsh, Bockus, \u0026amp; Chen, 1981) (G\u0026ouml;kerk\u0026uuml;\u0026ccedil;\u0026uuml;k, Tramier, \u0026amp; Bertolin, 2020).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe objective was to identify NSE forms originating from within the meteorite after shaking and fracturing the fragments inside the culture flask. Initially, the meteorite fragments were cultured under basal conditions and observed using an inverted microscope before proceeding with shaking. After confirming the absence of free material, vigorous and dry shaking was performed to strike the meteorite fragments against the walls of the culture flask.\u003c/p\u003e\n\u003cp\u003eIntermittent observations of all fragment boundaries were conducted using the inverted microscope until the expulsion of material from the meteorite\u0026rsquo;s interior was observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Once the expulsion occurred, the material initially emerged in an organized manner, exhibiting intense Brownian motion (Supplementary Video 2). Within the ferrous aggregate structures extracted from the interior of the meteorite, we observed nanometric dark punctate entities displaying a dynamic transition over time: initially, many exhibited pronounced movement, characterized by oscillatory vibrations and abrupt displacements, but as the observation progressed, an increasing number of these particles gradually ceased their motion. Despite losing mobility, these particles remained clearly distinguishable from the surrounding ferrous material, retaining their individual visibility and contrast within the matrix (Supplementary Video 3).\u003c/p\u003e\n\u003cp\u003eOver time, all observed particles reached a static state, appearing embedded within what visually resembled a structured ferrous sulfate aggregate, yet without fully merging into it. This progressive immobilization suggests a transition from a dispersed, dynamic phase to a structurally associated but still distinct state, likely governed by physicochemical interactions such as electrostatic forces, mineral phase transitions, or localized changes in solubility rather than autonomous biological activity. The contrast between the initially motile punctate features and their eventual stabilization, while maintaining their distinct appearance, highlights the need for further investigation into the mechanisms driving this transition. While some of these structures may share characteristics with the NSE described in this study, their behavior within the ferrous mineral matrix suggests that abiotic forces play a dominant role in their displacement and final stabilization. It subsequently diffused more freely, forming compact zones between the structures (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In this process, a Bundled Aggregate Form (RF-I) was generated from the material expelled from the meteorite\u0026rsquo;s interior (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). This structure closely resembled the NSE described in human blood but featured more mineralized and compact protective walls.\u003c/p\u003e\n\u003cp\u003eA key observation in the expelled ferrous material was the dynamic behavior of nanometric dark punctate entities, which initially exhibited rapid oscillatory motion and abrupt displacements. However, over time, a progressive reduction in their movement was observed, eventually leading to their complete immobilization while remaining distinguishable within the ferrous matrix. This transition raises important questions about the mechanisms governing their displacement and subsequent stabilization.\u003c/p\u003e\n\u003cp\u003eThe gradual cessation of movement suggests that these entities are subject to physicochemical interactions rather than autonomous biological activity. One plausible explanation is that their initial movement was driven by Brownian motion, influenced by interactions with surrounding fluid molecules. As the system reached equilibrium, the kinetic energy dissipated, leading to a decline in their mobility. Additionally, their integration into the ferrous material may result from electrostatic interactions or mineral adsorption, where attractive forces between the particles and the surrounding matrix progressively restricted their displacement until stabilization was achieved.\u003c/p\u003e\n\u003cp\u003eAnother possible mechanism is the formation of microstructured aggregates within the ferrous material, where particles become physically trapped in a denser, more structured mineral matrix over time. This would explain why they remain clearly visible and distinct from the surrounding material, despite no longer exhibiting independent movement. Furthermore, localized changes in solubility or ionic concentration may contribute to their immobilization, effectively anchoring them within the growing structure of the ferrous sulfate-like matrix.\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the motion and subsequent stabilization of these punctate entities could be explained by abiotic processes. However, further research is needed to determine whether these dynamics result purely from physicochemical mechanisms or if additional factors may be involved. The progressive immobilization of these entities, while maintaining their structural integrity, highlights the complexity of the interactions occurring within the expelled material and underscores the need for further studies to elucidate the exact mechanisms driving their dynamic behavior.\u003c/p\u003e\n\u003cp\u003eIn some cases, the expelled structures diffused as streams that varied in compactness (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), displaying a less intense Brownian motion rather than complete free Brownian movement. Instead, they formed a flow of particles within a surrounding medium, which appeared denser and more cohesive. Some of these streams remained stable for a period, while others eventually dispersed, as observed in previous cases (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), before aggregating and developing into a Bundled Aggregate Form (RF-I) (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe aggregates formed from the expelled structures also showed arborescent growths with color changes, eventually forming densely packed and uniformly colored structures (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Some structures strongly resembled a Protective Wall Form (RF-II), but with mineralized walls characteristic of their meteoritic origin (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn addition to forming aggregates, the structures expelled from the meteorite progressively adhered to the plastic strips generated by scraping the fragments against the culture flask walls (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). These adherent structures grew until they completely covered the plastic skeleton beneath them, preserving the original shape while continuing to grow on this foundation (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003cstrong\u003e-\u003c/strong\u003e (1) A structure resembling a Protective Wall Form (RF-II) with a well-defined mineralized layer (red arrow). (2) An NSE in the RF-II/RF-III transition stage (red arrow), showing strong interaction with adhered structures collected from the meteorite (green arrow). (3) The same structure as in image (2), but under higher magnification, providing more detail. (4) A detailed examination of the NSE in RF-II/RF-III transition stage reveals zones of activity, with microvesicles (4a) observed within regions between the more mineralized walls. (5) A higher magnification view of the active region, highlighting the appearance of microvesicles, as clearly visible in the inset (5a). (6) A view of the NSE Protective Wall Form (RF-II) structure, showing a color similar to the original meteorite material, along with interactions with aggregations of structures expelled from the meteorite (green arrows).\u003c/p\u003e\n\u003cp\u003eWhile in most cases the NSE structures exhibited a color very similar to the original meteorite fragment, we identified samples with an absolutely identical color (Figs. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Within these samples, growth of structures was observed that closely resembled those expelled from the meteorite after shaking and fragmenting. In these cases, there was no doubt that they originated from inside the meteorite (Figs. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e) and that there was growth occurring within the Mineralized Laminated-Fiber Structure (RF-II). This sample (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e) displayed significant activity, as evidenced by the genesis of a Bundled Aggregate Form (RF-I) (red arrow) originating from an aggregation.\u003c/p\u003e\n\u003cp\u003eResults with MTG Staining.\u003c/p\u003e\n\u003cp\u003eDifferent stages of the NSE were observed, and all structures were positively stained with MTG. This included both highly mineralized forms, such as the Protective Wall Form (RF-II) (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e), and newly formed structures like Bundled Aggregate Form (RF-I) (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e). The intensity of the staining was very pronounced, leaving no doubt about its positivity.\u003c/p\u003e\n\u003cp\u003eMorphologies resembling worm-like or coral-like structures were identified, which are also comparable to forms occasionally found in human blood (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e). The staining allowed clear differentiation of the most active regions within the samples (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e). In recently formed Bundled Aggregate Forms (RF-I) (Fig. \u003cspan class=\"InternalRef\"\u003e14\u003c/span\u003e), the constituent bundles were distinctly visible.\u003c/p\u003e\n\u003cp\u003eAll structural forms were successfully stained with MTG, even the early stages of bundle formation (Fig. \u003cspan class=\"InternalRef\"\u003e15\u003c/span\u003e). Given the simplicity of some of these structures, we focused on identifying Free Forms (FF). Culture medium was extracted, stained with MTG, and meticulously examined across the entire slide under the microscope, analyzing both the full extension and height of the liquid between the coverslip and slide. Free and motile forms were successfully identified (Figs. \u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThese Free Forms (FF) exhibited structured mobility, characterized by the presence of two or three internal rounded densities that dynamically reorganized within the structure. While their function remains unclear, further studies could explore whether they play a role in the retention or transport of genetic elements. This internal reorganization appeared to influence the overall displacement pattern, producing what can be described as oscillatory propulsive movement. At times, these internal densities generated brief linear displacements, whereas in other instances, they remained largely localized, displaying subtle vibrational shifts. Such behavior indicates that FF forms exhibit a self-organizing structural dynamic, potentially regulated by internal physicochemical interactions, facilitating the encapsulation and stabilization of ssDNA sequences under environmental conditions (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e16\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e17\u003c/span\u003e) (Supplementary Video 4).\u003c/p\u003e\n\u003cp\u003eNotably, in some observations, pairs of FF exhibited synchronized movement, displaying nearly identical velocities, trajectories, and stopping patterns. This phenomenon suggests the presence of an interaction mechanism between the FF, possibly mediated by electrostatic forces, fluid dynamics, or coordinated structural responses. The near-identical motion and synchronized halting behavior observed in these cases reinforce the notion that FF displacement is not solely dictated by external random forces but may involve intrinsic structural or physicochemical coupling between these entities. Further studies are needed to determine whether this coordination is an emergent property of their composition or an active process driven by self-organizing dynamics.\u003c/p\u003e\n\u003cp\u003eThe defining characteristics of the Free Forms (FF) include:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eSize: Typically, Free Forms (FF) measure approximately 3\u0026ndash;4 \u0026micro;m in length and 1.5\u0026ndash;1.8 \u0026micro;m in diameter. However, in some cases, when only a single internal density is observed and the interior is uniformly stained with MTG, the overall size can be as small as 1 \u0026micro;m. This variation suggests that FF may exist in different structural states, potentially reflecting different stages of formation, compaction, or functional adaptation.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eInternal Densities: Two or three actively moving, rounded internal densities measuring\u0026thinsp;~\u0026thinsp;0.7 \u0026micro;m in diameter.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMobility: Exhibited oscillatory propulsive movement, with internal density shifts modulating its overall displacement. At times, these internal reorganizations resulted in linear shifts, whereas in other cases, the forms remained relatively stable while displaying localized adjustments. Such behavior may be linked to an internal structural mechanism facilitating the retention and protection of genetic material.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eWall Properties: Some forms displayed greater elasticity, with flexible walls that facilitated enhanced mobility. Others appeared more rigid, with less flexible walls restricting motion.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMTG Staining: All Free Forms (FF) were positively stained with MTG, which may indicate an affinity between their membrane composition and the dye, rather than direct evidence of metabolic activity. The uniform staining of both the Free Forms (FF) and their internal condensations with MTG suggests a chemically homogeneous structure rather than a biologically compartmentalized system. This observation supports the hypothesis that these formations may be governed by physicochemical interactions rather than metabolic activity, reinforcing their potential role as structured compartments for the stabilization and transport of genetic elements.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThese findings demonstrate the structural dynamism and morphological diversity of the NSE, particularly the distinctive properties of the Free Forms (FF). Their internal density reorganization and observed mobility suggest a complex interplay of physicochemical forces, which could contribute to the stabilization of genetic or molecular components. However, the absence of clear metabolic activity or replication indicators prevents definitive classification as biological entities. Future work should focus on experimentally testing their ability to interact with nucleic acids, as well as exploring their formation under controlled laboratory conditions to better understand their origin and functional properties.\u003c/p\u003e\n\u003cp\u003eThe movement of Free Forms (FF) exhibits a distinct oscillatory pattern that does not conform to conventional biological motility mechanisms. While potential explanations include physicochemical factors such as localized surface tension differentials, asymmetric Brownian motion, or electrostatic interactions, additional studies are needed to determine the underlying causes. Future research employing high-speed tracking, microfluidic assays, and computational modeling will be crucial for clarifying whether this motion results from intrinsic structural properties or external environmental influences.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study utilized an innovative optical microscopy technique (LOL) to characterize and dynamically visualize Novel Structured Entities (NSE) observed in meteorite-derived cultures. The analysis revealed four distinct morphologies: Free Form (FF), Bundled Aggregate Form (RF-I), Protective Wall Form (RF-II), and Biofilm Form (BF), all exhibiting unique organizational features. These formations, consistently detected across meteorite samples from different classifications (ordinary and carbonaceous chondrites), suggest a structured but still poorly understood phenomenon. The observed structural reorganization, particularly under MTG staining, underscores their dynamic nature. While these findings open new avenues for further investigation, the precise nature of these structures\u0026mdash;whether they are abiotic self-assembled systems or biologically relevant entities\u0026mdash;remains to be determined. Their presence in meteorite-derived cultures makes them an intriguing subject for studies on molecular stability and organization in extreme environments.\u003c/p\u003e \u003cp\u003eThe identification of novel ssDNA sequences in meteorite-derived cultures introduces an additional genomic aspect to the study of NSE structures. Metagenomic analysis indicates that some of these sequences lack homologs in existing databases (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025), suggesting they may correspond to previously uncharacterized genetic elements. Certain sequences exhibit conserved motifs, distinct secondary structures, and a high AT-content (54.14% in MT_PURE sequences), features sometimes linked to adaptation to extreme environments.\u003c/p\u003e \u003cp\u003eWhile some extremophilic genetic elements, such as ssDNA viruses and plasmids (Krupovic, Dolja, \u0026amp; Kooni, 2019) (Labont\u0026eacute; \u0026amp; Suttle, 2015) exhibit similar traits, there is currently no evidence indicating any functional or direct association between these ssDNA sequences and NSE structures. Future studies should prioritize experimental validation through in situ hybridization, isotopic labeling, and the controlled synthesis of analogous structures to explore potential interactions or patterns of co-occurrence with NSE. Alternatively, these sequences could be independent elements within the culture medium. Further research is required to determine their potential structural or functional significance, if any.\u003c/p\u003e \u003cp\u003eFurthermore, the detection of shared sequence identities between the MT_TOTAL and MT_PURE datasets suggests that some of these sequences are highly conserved within the analyzed samples, reinforcing their structural and functional consistency across different meteorite-derived cultures. Notably, motif discovery analysis identified highly conserved repetitive elements that could serve as replication origins, regulatory regions, or structural domains. Given the absence of homologous sequences in known databases, future research should focus on in situ hybridization, isotopic labeling, and single-cell genomic techniques to determine whether these sequences are endogenous to NSE or originate from associated self-replicating genetic elements. Establishing a functional relationship between NSE and these ssDNA sequences will be crucial to understanding whether these structures serve as protective compartments for genetic material, facilitating its stabilization and potential replication under extreme conditions.\u003c/p\u003e \u003cp\u003eThe detection of structured ssDNA sequences in meteorite-derived cultures (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025) prompts further investigation into the stability and organization of nucleic acids in extreme environments. These sequences could hypothetically be related to prebiotic chemical processes or arise from abiotic self-assembly under specific physicochemical conditions. However, at this stage, their origin remains undetermined, and additional experimental validation is needed to discern between these possibilities. To explore these possibilities, future research should combine high-resolution molecular analysis, isotope labeling, and synthetic reconstruction of comparable structures to determine whether these sequences have prebiotic relevance or result from abiotic interactions. However, the association between these sequences and NSE structures remains highly speculative. Given the self-assembled nature of many complex physicochemical formations, it is plausible that NSE arise from abiotic self-organization processes, akin to mineralogical templating or vesicular compartmentalization observed in prebiotic chemistry. Future studies should focus on differentiating between self-organized abiotic structures and potential biological analogs, employing advanced imaging, isotopic tracing, and comparative analysis with known extremophilic systems to refine our understanding of these formations.\u003c/p\u003e \u003cp\u003eDifferential diagnosis of Free Forms (FF): a comprehensive analysis of possible candidates and insights from metagenomic data.\u003c/p\u003e \u003cp\u003eThe Free Forms (FF) identified in this study exhibit distinctive characteristics, including their dimensions (3\u0026ndash;4 \u0026times; 1.5\u0026ndash;1.8 \u0026micro;m), non-linear oscillatory displacement, dynamic internal densities (~\u0026thinsp;0.7 \u0026micro;m), and uniform positive staining with MTG. The mobility pattern of FF is markedly different from known microbial motility mechanisms. Unlike ultramicrobacteria, archaella-driven archaea, or magnetotactic bacteria\u0026mdash;which rely on external appendages, gliding, or magnetic alignment for movement (Lauga \u0026amp; Powers, 2009) (Blakemore, 1975) (Jarrell \u0026amp; McBride, The surprisingly diverse ways that prokaryotes move., 2008) \u0026mdash;FF display a motion that appears to originate from internal structural reorganization rather than active propulsion. This distinction suggests that their motility could be governed by physicochemical interactions, such as localized surface tension changes or electrostatic repulsion within the surrounding medium. Further high-resolution imaging and microfluidic assays will be essential to determine whether these movements result from biological processes or abiotic physicochemical mechanisms.\u003c/p\u003e \u003cp\u003eIn contrast, the FF display a unique irregularity in their motion, characterized by internal oscillations of their rounded densities, which drive abrupt transitions between stationary vibration and high-speed linear propulsion. This type of movement lacks parallels in known biological systems of similar dimensions. Magnetotactic bacteria, for instance, rely on static internal magnetosomes for navigation and produce a smooth helical trajectory under magnetic influence (Frankel, Bazylinski, Johnson, \u0026amp; Taylor, 1997 ) (Bazylinski \u0026amp; Frankel, 2004), while ultramicrobacteria, due to physical and energetic constraints associated with their diminutive size, are often non-motile or exhibit extremely limited motility (Dusenbery, 1997). Similarly, archaella-driven archaea use rotational mechanisms for propulsion but lack the dynamic internal reorganization observed in FF (Jarrell \u0026amp; McBride, The surprisingly diverse ways that prokaryotes move., 2008).\u003c/p\u003e \u003cp\u003eIn this study, we define this unique movement pattern as \u0026ldquo;oscillatory propulsive movement\u0026rdquo;, referring to the internally modulated shifts in density that result in periodic oscillations coupled with abrupt linear displacements. Unlike classical biological motility mechanisms such as flagellar rotation or gliding motility, oscillatory propulsive movement does not rely on external appendages or defined propulsion mechanisms. Instead, the movement appears to arise from intrinsic reorganizations within the structure, suggesting a physicochemical basis rather than an active biological motility system. Possible underlying mechanisms include localized surface tension variations, electrostatic interactions, or transient molecular rearrangements within the Free Forms (FF).\u003c/p\u003e \u003cp\u003eAdditionally, the irregular nature of oscillatory propulsive movement \u0026mdash;where FF alternate between high-speed bursts and localized oscillations\u0026mdash;contrasts sharply with random Brownian motion, which follows isotropic and thermodynamically driven patterns (Berg, 2018). Future studies employing high-speed video tracking, microfluidic assays, and computational modeling of fluid dynamics will be essential for characterizing the precise forces driving oscillatory propulsive movement and distinguishing it from biological motility systems. These experiments could also help determine whether the observed movement is modulated by external physicochemical gradients, such as ionic fluxes or energy differentials within the medium.\"*\u003c/p\u003e \u003cp\u003eThis behavior suggests the presence of a novel physicochemical mechanism for mobility and structural regulation, potentially influenced by internal density shifts, electrostatic interactions, or other non-biological forces adapted to extreme environments. The ability to shift between vibration and propulsion could represent an evolutionary strategy for resource acquisition, protection, or environmental adaptation. Furthermore, the combination of localized oscillations, high-speed propulsion, and flexible structural adaptations underscores the singularity of FF as a biological system. This dynamic internal movement may reflect a previously uncharacterized strategy for survival under harsh or resource-limited conditions, highlighting the need for further investigation using advanced imaging and molecular tools to elucidate its biological significance and underlying mechanisms.\u003c/p\u003e \u003cp\u003eThese features strongly suggest biological activity but raise questions about their classification and the need for a differential diagnosis. To address this, we compared FF with several bacterial and archaeal candidates based on their morphology, motility, and metabolic features (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Additionally, we integrated results from the metagenomic shotgun sequencing of the cultures, which provided insights into the microbial community composition and excluded many potential analogs.\u003c/p\u003e \u003cp\u003eNanoarchaea, such as Nanoarchaeum equitans, were considered as potential candidates due to their exceptionally small size (0.4\u0026ndash;0.5 \u0026micro;m), which overlaps with the dimensions of the internal rounded densities observed in FF (Waters, y otros, 2003). These archaea are obligate symbionts, typically associated with Ignicoccus species, from which they derive essential metabolites (Podar, Makarova, Graham, Koonin, \u0026amp; Reysenbach, 2013) While Nanoarchaea exhibit extreme genome reduction and metabolic simplicity, they lack the dynamic motility and flexible wall properties observed in FF. Additionally, Nanoarchaea do not possess the actively moving internal rounded densities characteristic of FF, and their strict dependence on a host for metabolic activity makes MTG staining unlikely. These key differences suggest that Nanoarchaea are an unsuitable analog for FF.\u003c/p\u003e \u003cp\u003eUltramicrobacteria, including species such as Pelagibacter ubique, represent another group of small, streamlined organisms that could theoretically share some minimalistic characteristics with FF. These bacteria are well-adapted to oligotrophic environments, with cell sizes typically ranging from 0.3 to 0.5 \u0026micro;m (Giovannoni, y otros, 2005). However, ultramicrobacteria are predominantly non-motile or, at most, exhibit limited flagellar motility. Importantly, they lack visible internal structures comparable to the actively moving rounded densities observed in FF (Lauro, y otros, 2009 ). Moreover, ultramicrobacteria possess highly constrained metabolic capacities, making it unlikely that they would exhibit positive MTG staining. These key differences further differentiate ultramicrobacteria from FF and suggest that they are not a suitable analog.\u003c/p\u003e \u003cp\u003eMagnetotactic bacteria, particularly species of the genus Magnetospirillum, share intriguing features with FF. These bacteria possess internal magnetosomes\u0026mdash;membrane-bound crystals of magnetite or greigite arranged in chains\u0026mdash;that enable alignment with magnetic fields (Barber-Zucker, Keren-Khadmy, \u0026amp; Zarivach, 2016 ). Magnetospirillum species are comparable in size, typically measuring 3\u0026ndash;5 \u0026micro;m in length and 0.5\u0026ndash;1 \u0026micro;m in width, and exhibit motility mediated by polar flagella (Zhang \u0026amp; Wu, 2020). However, their magnetosomes remain static and are arranged in linear chains, in contrast to the actively moving, rounded internal densities observed in FF. Furthermore, Magnetospirillum species exhibit a characteristic helical swimming pattern, driven by the rotation of their flagella, and do not display the oscillatory propulsive movement characteristic of FF (Reufer, y otros, 2014). Importantly, MTG staining is not typically associated with magnetotactic bacteria, as their energy generation and metabolic activity do not involve structures resembling mitochondria or related bioenergetic systems (Barber-Zucker, Keren-Khadmy, \u0026amp; Zarivach, 2016 ).\u003c/p\u003e \u003cp\u003eThe oscillatory movement observed in Free Forms (FF) differs from conventional microbial motility mechanisms involving external appendages, such as cilia, flagella, or gliding structures. While this movement appears to originate from internal structural dynamics, further research is necessary to determine whether it is governed by purely physicochemical forces or represents an alternative, yet uncharacterized, biological process.\u003c/p\u003e \u003cp\u003eAnother group considered was the Myxobacteria, a group of soil-dwelling bacteria known for their complex social behavior, biofilm formation, and swarming motility. While their ability to form organized aggregates may resemble the bundled forms (RF-I) observed in FF, Myxobacteria lack the dynamic individual motility displayed by FF (Cao, Dey, Vassallo, \u0026amp; Wall, 2015). Their swarming movement is collective, rather than individual, and they do not possess internal densities analogous to those seen in FF. Furthermore, the positive MTG staining observed in FF has not been reported in Myxobacteria, which further excludes them as candidates.\u003c/p\u003e \u003cp\u003eExtremophilic archaea, such as Halobacterium salinarum, were also analyzed due to their adaptability to extreme environments, a trait relevant to the origin of FF in meteorite-derived cultures. These archaea exhibit flagellar motility and are metabolically active under extreme conditions, which could theoretically explain MTG staining (Oren, 2002). However, their rigid cell walls, lack of internal rounded densities, and flagellar motility patterns (linear or rotational) do not align with the elastic wall properties and \u0026ldquo;oscillatory propulsive movement\u0026rdquo; motion of FF. Consequently, extremophilic archaea are also unlikely to be related to FF.\u003c/p\u003e \u003cp\u003eFinally, non-biological or prebiotic structures were evaluated as possible analogs. These include mineral inclusions or prebiotic aggregates that may form under specific chemical and physical conditions. Mineral inclusions can exhibit apparent mobility under microscopic observation due to Brownian motion or turbulence in liquid media (Barber \u0026amp; Scott, 2002 ) (Sephton, 2002) While these structures may superficially resemble FF, they lack key distinguishing properties, such as MTG staining, internally organized dynamics, and autonomous motility. These critical differences allow us to exclude non-biological structures as plausible explanations for the observed FF.\u003c/p\u003e \u003cp\u003eThe shotgun metagenomic analysis of the cultures revealed a taxonomically diverse microbial community, yet bacterial sequences constituted only 3% of the total DNA recovered. This relatively low bacterial presence makes it highly unlikely that widespread contamination could account for the observed Novel Biological Structures (NSE), including the Free Forms (FF). The taxonomic analysis identified Alphaproteobacteria as the most abundant group, particularly members of the Sphingomonadaceae family, with Sphingomonas paucimobilis (3%), Sphingomonas hankookensis (2%), and Sphingomonas alpina (1%) being the most prevalent species. These bacteria are known for their ability to survive in oligotrophic environments and metabolize complex organic compounds, suggesting a possible interaction with meteorite-derived materials.\u003c/p\u003e \u003cp\u003eAdditionally, other Alphaproteobacteria, such as Paracoccus and Erythrobacter (0.6%), were detected, along with members of Betaproteobacteria (Burkholderia), Firmicutes (Staphylococcus, Bacillus), and Actinomycetales (Streptomyces). Minor contributions from Acidobacteriota, Myxococcota, and the Terrabacteria group were also present, many of which are associated with resilience in extreme environments. Despite this microbial diversity, none of these taxa exhibit the combination of motility, self-organization, and structural characteristics observed in FF, further supporting the notion that the NSE are not attributable to known bacterial contaminants but represent an independent phenomenon within the cultures.For instance, Alphaproteobacteria and Betaproteobacteria typically lack the oscillatory propulsive movement seen in FF and instead rely on classical flagellar motility or passive dispersal (Newton, y otros, 2010) (Coenye \u0026amp; Vandamme, 2003). Firmicutes, such as Bacillus, are known for their ability to form spores, but their motility is flagella-driven and lacks the internal density oscillations characteristic of FF (Nicholson, Munakata, Horneck, Melosh, \u0026amp; Setlow, 2000). Actinomycetales, while capable of forming complex colonial structures, do not display individual motility nor dynamic internal features (Chandra \u0026amp; Chater, 2014 ).\u003c/p\u003e \u003cp\u003eThe metagenomic results suggest that the FF and other NSE do not easily align with any of the detected bacterial taxa, making it difficult to attribute their unique characteristics to known microorganisms. Additionally, the fact that a substantial portion of the sequences remained unclassified (51%) suggests the presence of unknown or highly divergent genetic elements, aligning with the previous discovery of novel single-stranded DNA (ssDNA) sequences in the same cultures. The combined molecular and morphological evidence suggests that the NSE, including the FF, may represent a previously uncharacterized structured system rather than artifacts of known microbial contaminants. Notably, the high percentage of unclassified sequences further supports the presence of unknown genetic elements. To elucidate their precise nature, future studies should focus on targeted molecular analyses to clarify the relationship between the FF structures and the detected ssDNA sequences.\u003c/p\u003e \u003cp\u003eWhile the structural characteristics and association with novel genetic elements are provocative, definitive conclusions regarding their biological or abiotic nature cannot yet be drawn. Additional experimental evidence is necessary to clarify these intriguing associations.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparative Summary Table\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCharacteristic\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFree Forms (FF)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNanoarchaea\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUltramicrobacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMagnetotactic Bacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMyxobacteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eExtremophilic Archaea\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNon-Biological\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSize (\u0026micro;m)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u0026ndash;4 \u0026times; 1.5\u0026ndash;1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.4\u0026ndash;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.3\u0026ndash;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3\u0026ndash;5 \u0026times; 0.5\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u0026ndash;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u0026ndash;5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMobility\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ldquo;oscillatory propulsive movement\u0026rdquo;, dynamic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLimited\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHelical, magnetic-field\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCoordinated swarming\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eFlagellar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eApparent (Brownian)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInternal Densities\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotile, rounded (0.7 \u0026micro;m)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStatic magnetosomes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMTG Staining\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePossible\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eElasticity of Wall\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRigid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRigid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRigid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRigid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRigid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEnvironment\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeteorite-derived cultures\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHost-dependent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOligotrophic waters\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAquatic sediments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTerrestrial soils\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eExtreme environments\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMeteorites, minerals\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOne of the striking features of the Free Forms (FF) observed in this study was their extreme rarity within the cultures. In an entire microscopic field using a 63\u0026times;/0.75 objective, typically five or fewer Free Forms (FF) were observed per slide, with a maximum of up to ten. This scarcity posed significant challenges for isolating and attempting to culture them, as well as for obtaining high-quality samples for electron microscopy analysis.\u003c/p\u003e \u003cp\u003eThe isolation of FF within the cultures is an important observation that argues against the possibility of contamination. In cases of contamination, microbial growth is typically abundant and widespread, producing clusters or biofilms that are readily visible under the microscope. In contrast, the FF were singular and sparsely distributed, further reinforcing the notion that they are intrinsic to the meteorite-derived cultures and not an artifact of external contamination. The data indicate that the NSE are unlikely to be the result of external contamination and may be intrinsic to the meteorite-derived cultures; however, further confirmation is required to rule out alternative explanations\u003c/p\u003e \u003cp\u003eAnother critical consideration in evaluating the nature of the Free Forms (FF) is the potential for environmental contamination. Microbial contaminants are ubiquitous in laboratory settings and can occasionally mimic unusual structures observed in culture systems. Among the most plausible candidates for contamination are bacteria like Bacillus spp., Pseudomonas spp., and Mycoplasma spp., as well as certain archaea such as halophiles or methanogens, all of which are common in diverse environments and capable of surviving under laboratory conditions. However, a detailed analysis of these potential contaminants highlights key differences that make it unlikely for any of them to explain the observed FF structures.\u003c/p\u003e \u003cp\u003eBacillus spp. are well-known environmental bacteria capable of forming resilient endospores (McKenney, Driks, \u0026amp; Eichenberger, 2013), which could superficially resemble the internal rounded densities observed in FF. Their size range, typically between 2 and 5 \u0026micro;m (Errington \u0026amp; Aart, 2020), aligns with the dimensions of the FF. However, endospores are static structures and lack the dynamic internal movement that is a defining feature of FF. Additionally, Bacillus species are motile via flagella (Mukherjee \u0026amp; Kearns, 2014)but exhibit linear or random motion rather than the \u0026ldquo;oscillatory propulsive movement\u0026rdquo;, highly variable movement observed in FF. Another critical point is that if Bacillus were responsible for the FF, they would likely be present in higher numbers across the cultures. Contamination by Bacillus typically results in visible clusters or widespread presence, yet FF were isolated and required meticulous scanning of the slide to locate a single structure. Moreover, the ability of Bacillus to form spores, a static survival structure, contradicts the active, motile nature of FF (Nicholson, Munakata, Horneck, Melosh, \u0026amp; Setlow, 2000) (Setlow, 2014).\u003c/p\u003e \u003cp\u003eSimilarly, Pseudomonas spp. are another group of ubiquitous environmental bacteria that are commonly associated with contamination in laboratory cultures. These bacteria are known for their metabolic versatility and active flagellar motility. While Pseudomonas species are smaller than FF, typically measuring 0.5 to 0.8 \u0026micro;m in width and 1.5 to 3.0 \u0026micro;m in length (Sampedro, Parales, Krell, \u0026amp; Hill, 2015), their high motility might seem superficially similar. However, Pseudomonas motility is driven by flagella (Bouteiller, y otros, 2021 )and is neither \u0026ldquo;oscillatory propulsive movement\u0026rdquo; nor characterized by the changes in speed and direction that define FF movement. Additionally, Pseudomonas do not exhibit any internal structures that could correspond to the rounded, motile densities of FF. More importantly, as opportunistic contaminants, Pseudomonas would proliferate rapidly in the culture media, leading to an abundance of cells that would be easily observable across the microscope slide. The isolated presence of FF, with only one structure observed per field at high magnification, is inconsistent with Pseudomonas contamination (Silby, Winstanley, Godfrey, Lev, \u0026amp; Jackson, 2011) (Silby, Winstanley, Godfrey, Lev, \u0026amp; Jackson, 2011).\u003c/p\u003e \u003cp\u003eMycoplasma spp., members of the Mollicutes class, represent another group of potential contaminants due to their small size and adaptability to diverse environments. These bacteria lack a cell wall, which gives them an elastic structure, potentially resembling the flexible walls of FF. Mycoplasmas are extremely small, typically 0.15\u0026ndash;0.3 \u0026micro;m in diameter (Nikfarjam \u0026amp; Farzaneh, 2012), significantly smaller than the 3\u0026ndash;4 \u0026micro;m size observed for FF (Kasai, y otros, 2013 ). They also lack visible internal structures and are non-motile, relying on gliding rather than \u0026ldquo;oscillatory propulsive movement\u0026rdquo; movement. While Mycoplasma species are notorious contaminants in cell culture systems, their rapid growth and dispersal in media would result in widespread contamination, making their presence highly abundant in any field of view. The rarity of FF is inconsistent with the contamination patterns of Mycoplasma (Razin, Yogev, \u0026amp; Naot, 1998) (Drexler \u0026amp; Uphoff, 2002).\u003c/p\u003e \u003cp\u003eAmong archaea, halophilic species such as Halobacterium spp. are another plausible candidate due to their adaptability to extreme conditions and potential contamination during culture preparation. Halophilic archaea range in size from 1 to 5 \u0026micro;m (Oren, 2002), overlapping with the dimensions of FF. They are metabolically active, and some species exhibit motility via archaella, which are functionally analogous to bacterial flagella. However, their motion is typically linear or rotational(Albers \u0026amp; Jarrell, 2015 ) rather than \u0026ldquo;oscillatory propulsive movement\u0026rdquo;, and they lack dynamic internal densities. Methanogenic archaea, such as Methanobrevibacter spp., also merit consideration. These microorganisms are slightly smaller, measuring 0.5\u0026ndash;2 \u0026micro;m (Liu \u0026amp; Whitman, 2008), and possess elastic cell walls that could be superficially similar to FF. However, methanogens are non-motile or exhibit minimal flagellar motility (Jarrell, Ding, Nair, \u0026amp; Siu, 2013 ), and their metabolic properties are incompatible with the intense MTG staining observed in FF. Furthermore, like bacterial contaminants, both halophiles and methanogens would likely appear in greater abundance if they were environmental contaminants, and their isolated presence on the slide argues against this explanation (Oren, 2002) (Garcia, Patel, \u0026amp; Ollivier, 2000) (Valentine, 2007).\u003c/p\u003e \u003cp\u003eEnvironmental contamination is typically characterized by high cell densities and widespread distribution, yet the FF were sparsely distributed and difficult to locate, with only one structure observed per field at 63\u0026times; magnification. This isolated distribution pattern, coupled with the unique morphological and dynamic features of FF, strongly argues against contamination as their source. The dynamic internal densities, \u0026ldquo;oscillatory propulsive movement\u0026rdquo; motility, and positive MTG staining further differentiate FF from common environmental microorganisms. These observations collectively support the hypothesis that FF may be intrinsic to the meteorite-derived cultures and not artifacts of laboratory contamination.\u003c/p\u003e \u003cp\u003eNevertheless, the possibility remains that the Free Forms (FF) could represent a contamination from an unknown extremophilic microorganism. The isolated and sparse nature of the FF, while arguing against traditional environmental contaminants, could also be consistent with the behavior of a highly specialized extremophile. These organisms are often adapted to extreme environments and may not proliferate abundantly under standard laboratory conditions, leading to their low representation in cultures. Extremophiles frequently have specific metabolic and ecological requirements that are difficult to replicate in laboratory media, which can result in their underrepresentation in experimental systems and contribute to the difficulty of their detection (Pham \u0026amp; Kim, 2012) (Koch, 1997). Furthermore, their unique characteristics, such as \u0026ldquo;oscillatory propulsive movement\u0026rdquo; motility, dynamic internal densities, and positive MTG staining, could reflect adaptations to environmental conditions that are not typically encountered in laboratory settings.\u003c/p\u003e \u003cp\u003eThe FF observed in this study exhibit traits reminiscent of extremophilic organisms known to thrive in Earth's most inhospitable environments. Halophilic archaea, for instance, possess specialized adaptations for survival in hypersaline environments, such as unique membrane lipids and motility via archaella, but their motion is typically linear or rotational rather than the dynamic \u0026ldquo;oscillatory propulsive movement\u0026rdquo; movement observed in FF (Oren, 2002). Similarly, thermophilic and acidophilic microorganisms, while robust and resilient to environmental stress, exhibit static structural characteristics that contrast with the flexible, dynamic features of FF. The isolation and rarity of FF in the cultures mirror the ecological behavior of extremophiles, which are often found in low abundance in their natural niches due to their highly specific environmental requirements. This raises the intriguing possibility that FF could represent a novel extremophilic structure or organism that has not yet been characterized.\u003c/p\u003e \u003cp\u003eWhile the nature and origin of the FF remain uncertain, their study provides an opportunity to expand our understanding of microbial diversity and the limits of structural adaptations. Further research integrating advanced genomic and proteomic analyses, as well as environmental simulations, will be essential to determine whether the FF represent an entirely novel biological structure or a previously uncharacterized extremophilic adaptation.\u003c/p\u003e \u003cp\u003eFurthermore, the presence of Free Forms (FF) in the same meteorite-derived cultures where novel ssDNA sequences were identified in a parallel study (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025) provides an intriguing possibility that these structures could be functionally related to the genetic elements detected. The ssDNA sequences found in these cultures, which lack similarity to known genomic databases, represent a groundbreaking discovery in themselves. The dynamic behavior, positive MTG staining, and structural uniqueness of the FF suggest that they could act as a genetic transport and stabilization system, providing a structured environment that preserves ssDNA sequences and potentially contributes to their persistence under extreme conditions. This raises the hypothesis that the FF may serve as genetic transport systemmanifestation or carrier of these sequences, offering a potential explanation for their functionality within these specialized cultures.\u003c/p\u003e \u003cp\u003eIf this connection is validated, it would represent a major advancement in understanding how novel genetic elements, such as the ssDNA sequences detected, might correspond to previously unknown biological structures. This highlights the importance of integrating morphological, molecular, and genomic data to explore the nature and role of the FF. Future studies aimed at directly linking the FF to these ssDNA sequences, through genomic localization, transcriptomic analyses, or proteomic studies, will be critical to confirm this functional relationship and further characterize their biological significance.\u003c/p\u003e \u003cp\u003eAnother key aspect of the findings in this study is the presence of distinct forms of the NSE in meteorite-derived cultures and their apparent similarities to those previously observed in human blood and amniotic membranes. The presence of these structures in vastly different contexts raises questions about their ubiquity, origin, and potential biological roles.\u003c/p\u003e \u003cp\u003eIn meteorite-derived cultures, several NSE forms were identified, including the Protective Wall Form (RF-II), Bundled Aggregate Form (RF-I), and Biofilm Form (BF). These structures exhibit morphological and staining similarities to those observed in human-derived samples. For instance, the RF-II \"worm-like\" and \"coral-like\" structures observed in meteorite cultures closely resemble analogous forms found in human blood samples. Similarly, the RF-I forms, characterized by their bundled appearance and association with dynamic vesicles, parallel the aggregative structures previously seen in amniotic membranes. Despite these similarities, there are notable differences that set the meteorite-derived NSE apart.\u003c/p\u003e \u003cp\u003eOne key difference is the degree of mineralization observed in the structures derived from meteorites. In particular, the RF-II forms found in the meteorite cultures exhibit a higher degree of rigidity and apparent mineral association, which could reflect adaptation to extraterrestrial conditions or the influence of the meteorite's mineralogical composition. This contrasts with the more flexible, biologically active RF-II forms found in human-derived samples (Supplementary Video 1). Furthermore, the association of the RF-I forms in meteorite cultures with vesicles containing unknown material raises additional questions about their biological significance. While vesicular structures have been observed in the RF-I forms in amniotic membranes, the vesicles in the meteorite-derived NSE could represent a distinct adaptation, potentially related to storage or transport of molecules under harsh environmental conditions.\u003c/p\u003e \u003cp\u003eThe presence of biofilm-like formations (BF) in meteorite-derived cultures also warrants particular attention. These structures, which exhibit pseudo-tissue-like appearances and compartmentalization, have a striking resemblance to biofilms observed in human blood. However, their sparse distribution and mineral inclusions in the meteorite samples suggest a potential functional or compositional divergence. The biofilm formations in meteorite-derived cultures might represent an adaptation to microgravity or extraterrestrial conditions, facilitating survival in nutrient-poor and physically extreme environments.\u003c/p\u003e \u003cp\u003eThe parallels and distinctions between the NSE forms observed in meteorite-derived cultures and those found in human-derived samples open intriguing questions about their origin and evolution. One possibility is that the meteorite-derived NSE could represent a convergent adaptation to environmental stressors, mirroring biological strategies observed in terrestrial systems. Alternatively, these structures might share a common origin or precursor, with their divergent features arising from environmental pressures unique to their respective contexts. The mineralized nature of the meteorite-derived NSE, coupled with their unique vesicular associations, supports the notion that these structures may have evolved in response to extraterrestrial conditions, providing a novel perspective on extremophilic adaptations.\u003c/p\u003e \u003cp\u003eFurther comparative studies between NSE forms in meteorite cultures and human-derived samples will be crucial for elucidating their shared and distinct features. These investigations could include advanced imaging techniques, compositional analyses, and genomic or proteomic studies to uncover potential evolutionary, structural, or functional connections between these fascinating structures.\u003c/p\u003e \u003cp\u003eThe fact that the flasks were observed twice daily using an inverted microscope provided valuable insights into the progression of structures present in the cultures from the very beginning and those that appeared later. This detailed monitoring allowed researchers to discern which NSE forms emerged during the frequent shaking of the flasks or through the reconfiguration of structures originating from the meteorite fragments. Consequently, this systematic observation confirmed that some of the NSE-like structures found during the experiment appeared as a result of the cultivation process and were not present initially as contamination associated with the meteorite fragments.\u003c/p\u003e \u003cp\u003eThe discovery of isolated Free Forms (FF) with unique characteristics and difficult taxonomic assignment led to the idea of exploring the \"no-hits\" region of the metagenomic shotgun sequencing data from these cultures. This effort aimed to identify novel or original ssDNA sequences that could potentially be associated with these distinctive motile forms. ssDNA was hypothesized as a potential source of genetic material in extreme environments due to its inherent resistance to harsh conditions and its adaptability, which has been demonstrated in certain viral and microbial systems where ssDNA plays a crucial role in survival and replication under stress (Gil, y otros, 2021) (de la Higuera \u0026amp; L\u0026aacute;zaro, 2022). Remarkably, this hypothesis was validated, as approximately 80% of the DNA extracted from these cultures was identified as ssDNA, a significant proportion of which represented novel genetic elements. These findings, detailed in a previously published study (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025), provided a molecular basis for understanding the FF and opened the door to further investigations into their biological nature and potential functionality.\u003c/p\u003e \u003cp\u003eThe discovery of novel ssDNA sequences in the same meteorite-derived cultures as the Free Forms (FF) and other observed forms of the NSE suggests a direct functional or structural association between these genetic elements and the biological structures identified. These ssDNA sequences, representing approximately 80% of the DNA extracted from these cultures, exhibit unique characteristics, such as AT-rich composition, conserved secondary structures, and repetitive motifs, which may indicate their role in replication, molecular regulation, or structural stabilization. The staining of all NSE forms, including the FF, Bundled Aggregate Form (RF-I), Protective Wall Form (RF-II), and Biofilm Form (BF), with MTG highlights metabolic or bioenergetic activity that could be linked to the functionality of these novel genetic elements.\u003c/p\u003e \u003cp\u003eThe association between these novel ssDNA sequences and the diverse morphologies of the NSE observed in this study raises intriguing questions about their potential roles in biological organization, replication, and adaptation. The FF, with their dynamic motility, internal rounded densities, and elastic walls, may represent the most active and motile expression of these genetic elements. Similarly, the more organized RF-I, RF-II, and BF forms could reflect stages of aggregation, adaptation, or interaction driven by these genetic components. The observation of these structures at different stages of culture development suggests that the ssDNA sequences may play a central role in driving the formation and behavior of the NSE.\u003c/p\u003e \u003cp\u003eThese findings propose a potential evolutionary and functional link between the ssDNA sequences and the NSE forms, highlighting their adaptability to the challenging conditions within the meteorite-derived cultures. The inherent stability and adaptability of ssDNA, as noted in studies of extremophilic microorganisms and viruses, suggest that these sequences could be well-suited to environments characterized by limited resources and extreme physical or chemical conditions (Cavicchioli, Siddiqui, Andrews, \u0026amp; Sowers, 2002 ) (Klein, 2020). This link emphasizes the importance of further investigation into the molecular and structural interplay between the ssDNA sequences and the NSE forms, as it could unveil novel strategies of survival and replication in extreme environments.\u003c/p\u003e \u003cp\u003eTo confirm this association, future studies should employ targeted genomic and proteomic approaches to localize the ssDNA within the NSE structures and identify potential proteins or other molecules mediating their interactions. Such studies could provide critical insights into the role of ssDNA in the organization and function of these biological forms, as well as their broader implications for extremophilic biology and the potential for novel life forms in extraterrestrial-like environments.\u003c/p\u003e \u003cp\u003eThe NSE observed in meteorite-derived cultures may represent structured, adaptive formations capable of encapsulating and vehiculizing single-stranded DNA (ssDNA). These structures appear to exhibit mechanisms for maintaining the integrity of ssDNA under diverse environmental conditions, potentially acting as protective molecular carriers rather than metabolically active entities. These structures, with their diverse morphologies and dynamic behaviors, could be expressions of the unique properties of ssDNA, which appears well-suited for survival and adaptation under a wide range of environmental conditions, including potentially extreme ones. The ssDNA sequences, characterized by their AT-rich composition, conserved motifs, and secondary structural stability, likely confer molecular flexibility and functional resilience, allowing these genetic elements to persist under conditions of resource scarcity or environmental stress.\u003c/p\u003e \u003cp\u003eThe structured mobility of Free Forms (FF) is best explained by physicochemical interactions within their internal architecture, rather than by known biological motility mechanisms. Their association with ssDNA sequences, previously identified in these same cultures, suggests that these formations could serve as structured compartments that facilitate the stabilization and possible retention of autoreplicative ssDNA sequences under extreme conditions.\u003c/p\u003e \u003cp\u003eThe proposed role of NSE as protective carriers of ssDNA finds parallels in known biological and synthetic systems that encapsulate and stabilize genetic material. For instance, ssDNA viruses such as Circoviridae and Parvoviridae utilize protein-based capsids to shield their genomes from enzymatic degradation and environmental stressors, ensuring their persistence and infectivity under extreme conditions (Delwart \u0026amp; Li, 2012). Similarly, extracellular vesicles in eukaryotic systems transport nucleic acids within lipid bilayers, protecting them from degradation in biological fluids (Mateescu, y otros, 2017). In nanomedicine, lipid nanoparticles (LNPs) have been extensively used to encapsulate mRNA and ssDNA for targeted delivery, demonstrating that structured compartments can play a crucial role in nucleic acid stabilization (Hou, Zaks, Langer, \u0026amp; Dong, 2021).\u003c/p\u003e \u003cp\u003eGiven the structural organization of NSE, it is plausible that they act as physicochemically stabilized compartments for ssDNA sequences, particularly under extreme environmental conditions. Their ability to transition between dynamic Free Forms (FF) and rigid Protective Wall Forms (RF-II) suggests an adaptive structural mechanism that could regulate the accessibility, transport, or preservation of genetic material. Future studies employing fluorescence in situ hybridization (FISH) or cryo-electron microscopy (Cryo-EM) could help determine the precise localization and interaction between NSE and their associated ssDNA, providing deeper insights into their structural and functional significance.\u003c/p\u003e \u003cp\u003eThe structural organization of NSE suggests a self-assembling system capable of dynamic morphological transitions, potentially enabling the stabilization and vehiculization of genetic material in response to environmental factors. The different NSE forms\u0026mdash;including Free Forms (FF), Bundled Aggregate Forms (RF-I), Protective Wall Forms (RF-II), and Biofilm Forms (BF)\u0026mdash;may reflect various strategies for encapsulating and safeguarding these genetic elements from environmental stressors. The more rigid and mineralized RF-II forms could act as external protective shells, ensuring stability, while the highly dynamic and motile FF may serve as active carriers for replication and dispersal. The NSE structures may also function as organized platforms facilitating replication, molecular protection, or metabolic activity, bridging molecular innovation with structural adaptation.\u003c/p\u003e \u003cp\u003eThese findings indicate that ssDNA sequences are associated with adaptable NSE structures, suggesting a potential evolutionary mechanism that may facilitate survival under extreme conditions. The co-occurrence of these ssDNA sequences with NSE in meteorite-derived cultures points to a possible role in extremophilic adaptations. While these observations raise the possibility of novel biological interactions and adaptive strategies, further research is needed to fully determine their significance in astrobiology and prebiotic molecular evolution.\u003c/p\u003e \u003cp\u003eThe observed adaptability and structural organization of the NSE suggest intriguing parallels with prebiotic molecular assemblies, particularly protocells, coacervates, and vesicle-like systems. These self-organizing structures, widely studied in the context of the origins of life, exhibit compartmentalization, selective permeability, and dynamic interactions with their surroundings\u0026mdash;features that are reminiscent of NSE behavior (Adamala \u0026amp; Szostak, 2013 ) (Chen \u0026amp; Walde, 2010). The ability of NSE to form aggregates, transition between different morphological states, and potentially interact with genetic material aligns with prebiotic models wherein simple molecular assemblies provided a framework for primitive biochemical processes.\u003c/p\u003e \u003cp\u003eNotably, protocell research has demonstrated that phase-separated compartments such as coacervates can accumulate, protect, and facilitate the replication of nucleic acids, creating conditions favorable for molecular evolution (Drobot, y otros, 2018 ) (Jia, y otros, 2019). The presence of structured ssDNA sequences within NSE suggests a similar function, where the compartmentalization of genetic material within adaptable biological structures might enhance molecular stability and persistence. This hypothesis aligns with previous studies on the role of membrane-free compartments in concentrating ribonucleotides and promoting early polymerization events, which could have driven the transition from chemistry to biology (Koga, Williams, Perriman, \u0026amp; Mann, 2011 ) (Patel, Percivalle, Ritson, Duffy, \u0026amp; Sutherland, 2015).\u003c/p\u003e \u003cp\u003eFurthermore, the role of meteorites in prebiotic chemistry is well-documented, with carbonaceous chondrites containing nucleobases, amino acids, and amphiphilic compounds capable of forming vesicle-like structures under appropriate conditions (Pearce, Pudritz, Semenov, \u0026amp; Henning, 2017) (Ferus, y otros, 2017 ). If the NSE observed in meteorite-derived cultures represent structures capable of molecular stabilization, replication, or selective chemical interactions, they may serve as experimental models for studying protocell-like behavior in extraterrestrial contexts. Given the environmental conditions under which these forms emerged, future studies should explore their potential stability in conditions mimicking early Earth or extraterrestrial environments, such as cycles of dehydration-rehydration, radiation exposure, and extreme temperature shifts (Rajamani, y otros, 2008) (de la Escosura, 2019).\u003c/p\u003e \u003cp\u003eBy drawing a more explicit comparison between NSE and prebiotic molecular assemblies, this study contributes to the growing body of research investigating the transition from non-living to living systems. If further investigations confirm that NSE function as primitive carriers of genetic information or as structurally adaptive molecular systems, they may represent a biological\u0026ndash;prebiotic continuum, bridging the gap between self-assembling molecular structures and the earliest forms of cellular organization. This perspective has profound implications for astrobiology, as it suggests that similar molecular systems could emerge and persist in diverse planetary environments, including Mars, Europa, and Enceladus, where conditions favor the preservation of organic material and the emergence of molecular complexity (Sutherland, 2017) (Jordan, y otros, 2019 ).\u003c/p\u003e \u003cp\u003eThe presence of nitrogen-rich soluble organic matter in pristine samples from asteroid Bennu reinforces the notion that carbonaceous asteroids could have served as reservoirs for prebiotic chemistry. Recent studies have identified amino acids, polycyclic aromatic hydrocarbons, and nitrogen-containing heterocycles\u0026mdash;including all five canonical nucleobases\u0026mdash;in Bennu\u0026rsquo;s organic inventory, suggesting that these bodies contained the fundamental building blocks necessary for life (Glavin, Dworkin, Alexander, JC, \u0026amp; al.., 2025). These findings align with our observations of microscopic structures within meteorite-derived cultures that appear to exhibit self-organization, resilience, and interaction with selective biological stains, including MTG. The ability of these structures to retain dye suggests the presence of lipid-like components or membrane-like formations, which could indicate a form of compartmentalization, a key feature in early prebiotic evolution. Given that Bennu samples contain high concentrations of ammonia and nitrogenous compounds\u0026mdash;both essential for amino acid and nucleotide synthesis\u0026mdash;it is plausible that similar organic reservoirs in the early Solar System contributed to the formation of protocellular structures.\u003c/p\u003e \u003cp\u003eFurthermore, the identification of NSE in our samples, potentially interacting with DNA probes, could indicate the presence of nucleotide-like components or their precursors. If these structures are indeed capable of binding nucleic acids, their formation in meteorite-enriched media may represent an experimental analog to early Earth conditions, where similar interactions could have contributed to the emergence of primitive genetic systems. The presence of racemic amino acids in Bennu samples further supports an abiotic origin, as no clear enantiomeric excess was detected (Glavin, Dworkin, Alexander, JC, \u0026amp; al.., 2025), reinforcing the idea that the organic inventory of meteorites represents a prebiotic rather than biological signature.\u003c/p\u003e \u003cp\u003eOur findings indicate that meteorite samples subjected to aqueous conditions give rise to dynamic and organized microstructures. This observation is consistent with the presence of evaporitic minerals in Bennu samples, which suggest prolonged interaction with liquid water in its parent body (McCoy, y otros, 2025). The detection of sodium-rich phosphates, carbonates, and sulfates in Bennu\u0026rsquo;s composition supports the idea that aqueous alteration may have facilitated organic molecule stabilization and catalytic processes relevant to early biochemistry. In our study, the ability of meteorite-derived samples to form ordered assemblies in aqueous media suggests that certain minerals or organics within the meteorite may act as catalysts or scaffolds, promoting molecular self-assembly.\u003c/p\u003e \u003cp\u003eThe correlation between self-organizing structures in our study and the presence of brine-derived salts in Bennu is particularly relevant. It has been suggested that phosphate-rich environments within asteroid parent bodies could have enabled phosphorylation reactions essential for nucleotide formation (McCoy, y otros, 2025). In our case, if the NSE observed in our cultures are interacting with nucleic acids or their precursors, the meteorite material may be providing a similar chemically enriched microenvironment, where prebiotic polymerization reactions could be favored. Additionally, Bennu\u0026rsquo;s brine chemistry, which shares similarities with subsurface oceans in icy bodies such as Ceres and Enceladus, reinforces the idea that transient but chemically rich environments could support molecular complexity and potentially act as crucibles for abiogenesis.\u003c/p\u003e \u003cp\u003eThese results provide compelling empirical support for the idea that prebiotic chemical evolution is not limited to controlled laboratory settings but can emerge in naturally occurring extraterrestrial environments. The simultaneous presence of nitrogen-rich organics and aqueous-altered minerals in Bennu samples (Glavin, Dworkin, Alexander, JC, \u0026amp; al.., 2025) (McCoy, y otros, 2025) strengthens the argument that meteorites could serve as both molecular reservoirs and reaction environments for self-organizing structures. If, in our study, meteorite-derived material can spontaneously give rise to protocell-like formations, it follows that similar processes may have occurred on early Earth or in other planetary bodies where liquid water was transiently or persistently available. When considered alongside the chemical complexity identified in Bennu, our findings suggest that carbonaceous asteroids could have played a direct role in fostering prebiotic systems, bridging the gap between chemistry and early biology.\u003c/p\u003e \u003cp\u003eComparison with known biological systems: potential parallels to the NSE hypothesis.\u003c/p\u003e \u003cp\u003eIf the hypothesis that the NSE are physical, functional, and protective manifestations of ssDNA sequences is correct, it would align with several known biological systems that employ similar strategies to protect, replicate, and maintain genetic material in extreme environments. These comparisons help contextualize the potential role of the NSE in the broader landscape of biological evolution and adaptation.\u003c/p\u003e \u003cp\u003e1. Plasmids and ssDNA viruses: genetic elements with protective mechanisms.\u003c/p\u003e \u003cp\u003eOne of the closest biological parallels to the hypothesized NSE system is the family of ssDNA viruses and rolling-circle plasmids, which have evolved highly specialized strategies to protect and propagate their genetic material. Viruses such as Circoviridae and Parvoviridae encapsulate their ssDNA genomes within highly stable protein capsids that shield them from enzymatic degradation and environmental stressors (Delwart \u0026amp; Li, 2012). Similarly, rolling-circle replication (RCR) is a mechanism employed by certain bacterial and archaeal plasmids, enabling rapid replication while maintaining structural integrity in challenging environments (Khan, 1997 ). This replication strategy allows for the continuous amplification of small ssDNA molecules, a feature that could be relevant if the NSE is indeed a biological system that relies on self-replicating genetic elements.\u003c/p\u003e \u003cp\u003eThe ability of these ssDNA elements to integrate into host genomes or persist as episomal entities (Desfarges \u0026amp; Ciuffi, 2012) suggests a potential functional link to the observed NSE forms. If the NSE structures are indeed harboring or facilitating the replication of ssDNA, they may represent a previously uncharacterized strategy for genetic persistence in extreme environments. The presence of dynamic, vesicle-like formations within the NSE structures could further support this idea, as these features resemble the proteinaceous compartments formed by certain ssDNA viruses to compartmentalize replication processes (Kazlauskas, Varsani, Koonin, \u0026amp; Krupovic, 2019).\u003c/p\u003e \u003cp\u003e2. Nanobacteria and vesicles as protective genetic compartments.\u003c/p\u003e \u003cp\u003eThe hypothesis that the NSE structures serve as protective environments for ssDNA also aligns with historical claims about nanobacteria\u0026mdash;submicron-sized entities that were once proposed as living microorganisms but are now more commonly regarded as self-organizing mineralized vesicles capable of enclosing genetic or biochemical material (Kajander \u0026amp; Cift\u0026ccedil;ioglu, 1998). These vesicles have been observed in extreme conditions, including mineral deposits and biological fluids, where they appear to form protective shells that could safeguard nucleic acids from degradation (C\u0026iacute;ft\u0026ccedil;\u0026iacute;oglu, Miller-Hjelle, Hjelle, \u0026amp; Kajander, 2002).\u003c/p\u003e \u003cp\u003eWhile the existence of true nanobacteria remains controversial, the fundamental principle behind their formation\u0026mdash;self-assembled nanoscale compartments that could house genetic material\u0026mdash;resonates with the observed morphologies of the NSE. The presence of mineral-associated forms, such as the Protective Wall Forms (RF-II), suggests that some NSE structures may engage in a similar process of encapsulation, potentially stabilizing the ssDNA within protective layers. This would allow the genetic elements to persist in harsh conditions, much like mineralized vesicles have been proposed to do in various biological and non-biological contexts (Wu, y otros, 2016).\u003c/p\u003e \u003cp\u003e3. Early endosymbionts and protocells: evolutionary precursors to cellular life.\u003c/p\u003e \u003cp\u003ePerhaps the most intriguing parallel to the NSE hypothesis comes from theories on the origin of life, particularly the role of protocells in the transition from self-replicating genetic elements to fully functional biological cells. Protocells are hypothesized to have been primitive vesicular structures that encapsulated genetic material, providing a stable microenvironment for replication and biochemical reactions (Szostak, 2012). The ability of lipid vesicles or mineral membranes to spontaneously form around nucleic acids has been demonstrated in various prebiotic chemistry experiments, suggesting that such structures could have been crucial in early molecular evolution (Maurer, Deamer, Boncella, \u0026amp; Monnard, 2009).\u003c/p\u003e \u003cp\u003eIf the NSE forms are indeed acting as a protective and functional system for ssDNA, they may represent an example of a naturally occurring, non-cellular genetic persistence mechanism. This could have implications for understanding the fundamental requirements for genetic continuity in extreme environments, as well as for exploring alternative models of biological organization beyond traditional cellular life. The observation that the NSE structures can exist in various morphologies\u0026mdash;ranging from dynamic Free Forms (FF) to organized biofilms (BF)\u0026mdash;may indicate an adaptive strategy similar to that seen in protocellular evolution, where different forms emerged in response to environmental pressures.\u003c/p\u003e \u003cp\u003eImplications and future investigations.\u003c/p\u003e \u003cp\u003eThe similarities between the NSE structures and these established biological systems suggest that the observed formations in meteorite-derived cultures could represent a novel genetic adaptation strategy, integrating protective, replicative, and survival mechanisms. If these structures indeed function as protective compartments for ssDNA, they may provide valuable insights into how genetic material can persist outside of conventional cellular frameworks.\u003c/p\u003e \u003cp\u003eTo further validate these comparisons, future research should aim to:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eDetermine the biochemical composition of NSE protective layers to assess their similarity to viral capsids, nanovesicles, or protocellular membranes.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eIdentify replication-associated proteins or motifs within the ssDNA sequences that may indicate functional parallels to rolling-circle replication or viral replication systems.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eInvestigate the self-assembly properties of the NSE structures to determine if they exhibit characteristics analogous to prebiotic vesicle formation.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eBy exploring these aspects, we may gain deeper insights into whether the NSE represents an unknown form of biological organization, an ancient survival strategy, or even a model for the potential persistence of genetic material in extraterrestrial environments.\u003c/p\u003e \u003cp\u003eThe findings of this study on NSE share intriguing parallels with the recent discovery of nematodes revived from Siberian permafrost after 46,000 years of cryptobiosis (Shatilovich, y otros, 2023 ). Both highlight the extraordinary capacity of biological systems to adapt and persist under extreme conditions. While the nematodes rely on specialized metabolic pathways, such as the synthesis of trehalose and the glyoxylate cycle, to endure desiccation and freezing, the NSE observed in meteorite-derived cultures may represent simpler systems, potentially centered around ssDNA sequences. The resistant forms of NSE, such as the Protective Wall Forms (RF-II) and Biofilm Forms (BF), could serve a function analogous to the cryptobiotic state in nematodes, providing structural protection and stability for the ssDNA under harsh conditions. On the other hand, the dynamic Free Forms (FF), with their \u0026ldquo;oscillatory propulsive movement\u0026rdquo; motility and motile internal densities, suggest active roles in replication or dispersal, possibly reflecting an adaptive strategy distinct from the dormancy observed in nematodes. Despite these functional differences, both systems underscore the possibility of long-term survival and reactivation of biological entities in extreme or extraterrestrial environments. This parallel raises fascinating questions about the evolutionary mechanisms underpinning resilience and adaptation across vastly different biological systems, offering a unique framework for exploring the limits of life on Earth and beyond.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrobiological implications of NSE and their association with ssDNA.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe discovery of NSE in meteorite-derived cultures, particularly their structural complexity and interaction with ssDNA sequences, raises questions about the potential role of self-organizing molecular systems in prebiotic chemistry. The morphological diversity of NSE, ranging from dynamic Free Forms (FF) to highly structured Protective Wall Forms (RF-II) and Biofilm Forms (BF), suggests a capacity for environmental adaptation, although the underlying mechanisms remain to be determined. Notably, the structural features of RF-II, which exhibit mineralized-like protective shells, may play a role in shielding nucleic acids or other biomolecules from environmental stressors such as desiccation or radiation, conditions that are dominant in many extraterrestrial environments, including Mars and the icy moons of Jupiter and Saturn (Cockell, 2014). While these properties resemble survival strategies observed in extremophilic microorganisms, further analysis is required to establish their functional significance in prebiotic or astrobiological contexts (Rothschild \u0026amp; Mancinelli, 2001).\u003c/p\u003e \u003cp\u003eThe presence of ssDNA, constituting nearly 80% of the extracted genetic material from these cultures, further highlights the need for a deeper understanding of the molecular composition and origins of NSE. Single-stranded DNA is known for its structural flexibility and ability to form stable secondary structures under stress conditions, raising the possibility that such molecules could contribute to stability or biochemical interactions within these self-organizing systems (Pal \u0026amp; Levy, 2019). However, the absence of close matches in genomic databases for the ssDNA sequences identified in this study underscores the importance of additional sequencing and comparative analyses before drawing conclusions about their evolutionary or environmental adaptations. The role of ssDNA in extremophilic microorganisms and viruses adapted to harsh conditions, including high radiation and resource scarcity, suggests that further investigation could reveal whether similar molecular mechanisms are at play in NSE-like systems (Pietil\u0026auml;, Roine, Paulin, Kalkkinen, \u0026amp; Bamford, 2009).\u003c/p\u003e \u003cp\u003eGiven that extremophiles on Earth employ diverse molecular strategies to withstand extreme environments, further investigation into NSE and their interaction with ssDNA may provide insights into how self-organizing systems emerge and persist under conditions relevant to early Earth or extraterrestrial environments. While these findings do not imply a direct link to extraterrestrial life, they underscore the importance of exploring meteorite-derived materials as experimental platforms for studying molecular self-assembly and stability in simulated planetary conditions. The possibility that extremophilic molecular strategies observed on Earth could reflect evolutionary pathways suitable for survival in extraterrestrial environments remains an open question in astrobiology (Chyba \u0026amp; Hand, 2005).\u003c/p\u003e \u003cp\u003eCosmic perspective: meteorites as carriers of ancient life or prebiotic molecules.\u003c/p\u003e \u003cp\u003eThe meteorite origins of the studied NSE introduce the intriguing possibility that these structures represent biological remnants or prebiotic systems preserved from extraterrestrial environments. Meteorites are well-documented carriers of organic molecules, including amino acids, nucleobases, and lipid precursors, which form the foundational building blocks of life (Pearce, Pudritz, Semenov, \u0026amp; Henning, 2017). Furthermore, the preservation of these molecules in meteorites, despite their exposure to cosmic radiation and the harsh interstellar medium, suggests that protective encapsulation mechanisms\u0026mdash;such as those potentially mirrored in RF-II and BF forms\u0026mdash;could enable the persistence of biological systems during space travel and planetary impact (Pizzarello, 2006).\u003c/p\u003e \u003cp\u003eThe ability of the NSE to \"reactivate\" under laboratory conditions, forming metabolically active and motile structures, raises parallels with long-term biological dormancy and cryptobiosis observed in Earth systems (Horneck, y otros, 2008). This capacity for reactivation under favorable conditions suggests a potential mechanism for persistence in fluctuating or extreme environments. While the direct relationship between these structures and extraterrestrial conditions remains to be determined, the presence of ssDNA and dynamic biological structures in meteorite-derived cultures warrants further investigation. These findings underscore the need for integrative approaches that combine molecular, structural, and astrobiological studies to assess potential biosignatures in planetary materials, complementing efforts in missions such as the Mars Sample Return program.\u003c/p\u003e \u003cp\u003eThe concept of \"Live Panspermia,\" as introduced in the study of ssDNA sequences in meteorite-derived cultures (L\u0026oacute;pez Ram\u0026oacute;n y Cajal, 2025), presents a compelling framework for understanding the biological and molecular systems potentially harbored in extraterrestrial materials. This notion posits that certain genetic elements, such as the novel ssDNA sequences identified, may have originated or been preserved in meteorites, carrying the potential for reactivation under favorable environmental conditions. The morphological and functional attributes of the NSE observed in this study align closely with the foundational ideas of Live Panspermia. Specifically, the reactivation of metabolically active, motile NSE forms under laboratory conditions mirrors the hypothesized ability of genetic systems to remain dormant during interstellar transit and subsequently regain activity when exposed to suitable conditions, such as hydration and nutrient availability. This connection reinforces the idea that NSE forms, including the Free Forms (FF) and protective wall structures, may represent physical manifestations of these genetic systems, adapted for survival in extreme or extraterrestrial environments. By linking the structural and molecular findings, this study contributes to the broader understanding of how meteorite-derived systems could inform theories on the dissemination and resilience of life across planetary boundaries.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProposed functional hypothesis of NSE and ssDNA dynamics.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe observations presented in this study, alongside the genomic evidence of abundant ssDNA sequences, suggest a potential model for how NSE structures adapt and respond to environmental conditions. The Free Forms (FF) may represent short ssDNA sequences in an active state, capable of dynamic motility and interaction with their surroundings. In response to environmental cues, such as changes in nutrient availability, osmotic pressure, or other external stresses, these ssDNA sequences could reorganize or fuse, leading to the development of protective walls and more complex structures, such as Bundled Aggregate Forms (RF-I) and Protective Wall Forms (RF-II). The RF-I forms, characterized by their less mineralized and more flexible structures, may facilitate environmental interaction or proliferation within the medium. By contrast, the RF-II forms, with their highly mineralized and compact walls, appear to prioritize long-term survival under harsh conditions by offering a high degree of protection.\u003c/p\u003e \u003cp\u003eIn these quiescent states, the ssDNA sequences and their associated structures likely enter a dormant phase, where their metabolic activity, if present, is minimal, and their extraction becomes challenging. This dynamic system of structural reconfiguration, oscillating between active motile states and quiescent protective forms, represents a previously uncharacterized adaptive structural mechanism for balancing environmental interaction, stabilization, and persistence. Unlike traditional microbial systems, the ability of ssDNA-driven NSE to transition seamlessly between phases may provide a novel mechanism for genetic material preservation, particularly in extreme or resource-limited environments. This hypothesis underscores the potential significance of ssDNA as not just a genetic element but a driver of structural and physicochemical adaptability, warranting further investigation using advanced imaging and molecular techniques.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe discovery and characterization of NSE in meteorite-derived cultures suggest that these structures may incorporate a biological component, exhibiting unique structural adaptations that could be relevant to extremophilic biology. We observed NSE in several morphologies, including Free Forms (FF), Bundled Aggregate Forms (RF-I), Protective Wall Forms (RF-II), and Biofilm Forms (BF), all of which showed intense MTG staining. Because MTG is typically associated with bioenergetic activity, these observations raise the possibility that these structures may perform active biological functions; however, further investigation is required to definitively distinguish them from abiotic aggregates.\u003c/p\u003e\n\u003cp\u003eThe dynamic motility, flexible structural adaptations, and well-organized aggregations of these forms differentiate them from known bacterial and archaeal candidates such as ultramicrobacteria, magnetotactic bacteria, Myxobacteria, and extremophilic archaea. Moreover, metagenomic shotgun sequencing of the cultures did not identify any known microorganisms that could account for the NSE, reinforcing the idea that these structures may represent a novel biological system. The absence of contamination patterns further supports the notion that the NSE are intrinsic to the meteorite-derived cultures rather than being laboratory artifacts.\u003c/p\u003e\n\u003cp\u003eA notable breakthrough was the identification of novel ssDNA sequences within the approximately 80% fraction of extracted ssDNA from the meteorite cultures. Only a subset of these sequences—originating from the no-hits region of the genomic analysis—was found to be novel (López Ramón y Cajal, 2025). These novel sequences exhibit unique AT-rich compositions, conserved motifs, and distinct secondary structural formations that suggest a high degree of adaptability. Their presence in these cultures raises the hypothesis that the various NSE morphologies may represent physical, functional, and protective manifestations of these ssDNA sequences. For example, the FF, with their “oscillatory propulsive movement” and dynamic internal densities, could function as motile carriers or dispersal forms of the ssDNA, while the more structured RF-I, RF-II, and BF forms may provide protective and adaptive environments conducive to replication and persistence.\u003c/p\u003e\n\u003cp\u003eThe observed association between FF and ssDNA sequences implies a potential interaction between structured compartments and genetic elements capable of autoreplication. Although the current findings do not demonstrate conventional biological activity, they offer new perspectives on how genetic material might persist and propagate under extreme conditions. This proposed link draws parallels with mechanisms such as rolling-circle replication in plasmids, encapsulation in ssDNA viruses (e.g., Circoviridae, Parvoviridae), and vesicle-like structures observed in nanobacteria. If validated, this model would describe a previously uncharacterized structured genetic system in which ssDNA elements dynamically transition between free, motile forms and structured protective aggregates, facilitating stabilization and persistence in harsh environments.\u003c/p\u003e\n\u003cp\u003eThese findings expand our understanding of microbial diversity and structural adaptation in extraterrestrial-like settings. The association of ssDNA sequences with NSE suggests a novel strategy for genetic persistence, structural organization, and bioenergetic function with potential implications for extremophile studies and the search for extraterrestrial life. Future research should focus on direct molecular localization of ssDNA within NSE structures, as well as transcriptomic and proteomic analyses and environmental simulations to further clarify the functional and evolutionary implications of this novel system. The integration of these approaches will be essential to determine whether NSE represent a previously unrecognized extremophilic adaptation or an entirely novel biological paradigm.\u003c/p\u003e\n\u003cp\u003eIn summary, the identification of metabolically active and structurally distinct NSE in meteorite-derived cultures provides a unique framework for understanding potential extraterrestrial life forms or prebiotic systems. By linking the observed morphologies to novel ssDNA sequences, this research proposes new mechanisms for genetic preservation and structural adaptation under extreme conditions. Although direct evidence linking NSE to extraterrestrial origins remains to be established, the methodologies employed in this study—including advanced microscopy and genomic analysis—offer a valuable framework for investigating biosignatures in planetary materials. These findings underscore the importance of integrating molecular, structural, and functional evidence to explore the potential diversity and resilience of life beyond Earth.\u003c/p\u003e\n\u003cp\u003eFinally, the NSE structures observed in this study may represent a previously uncharacterized framework that sheds light on the stability and persistence of genetic material under extreme conditions. Their dynamic behavior, morphological diversity, and association with novel ssDNA sequences highlight their potential as a model for understanding how genetic components can be stabilized, transported, and preserved in environments that challenge conventional biological frameworks. This study emphasizes the need for an open perspective in the search for life, suggesting that early forms of life may manifest as dynamic, adaptable, and structurally simple systems. In doing so, it invites further investigation and replication by multiple research groups to fully assess the significance and universality of these observations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations of the study.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough our findings offer intriguing insights into the nature of NSE, several limitations warrant cautious interpretation and further investigation. First, the microscopic characterization was limited by the resolution constraints of optical microscopy, which restricted our ability to perform a more detailed ultrastructural analysis. While MTG staining provided data consistent with metabolic activity, the exact biochemical composition and functional role of these structures remain to be clarified through additional spectroscopic and molecular approaches.\u003c/p\u003e\n\u003cp\u003eMoreover, although metagenomic shotgun sequencing identified a substantial number of novel ssDNA sequences, we were unable to directly localize these sequences within the NSE structures, leaving questions about their functional integration open. The challenge of culturing or isolating Free Forms (FF) in sufficient quantities further limited the scope of controlled experimental assays to evaluate their biological properties. Additionally, the extremely low density of NSE in the cultures necessitated extensive scanning and limited the material available for comprehensive analysis, highlighting the need for more advanced enrichment techniques.\u003c/p\u003e\n\u003cp\u003eFinally, despite rigorous efforts to rule out contamination, the possibility that these structures may originate from an unidentified environmental extremophile cannot be entirely excluded. Given these limitations, we emphasize the importance of independent replication by multiple research groups to validate and extend these findings, which will be critical for confirming the functional and biological significance of NSE.\u003c/p\u003e\n\u003cp\u003eFinally, despite rigorous efforts to exclude contamination, we cannot entirely rule out the possibility that NSE originate from an unidentified terrestrial environmental extremophile or physicochemical processes intrinsic to the culture conditions themselves. Although MTG staining produced results consistent with metabolic activity, such staining alone does not conclusively indicate active metabolism or biological function. The precise biochemical composition, functional significance, and potential biological nature of NSE remain undetermined and require further detailed molecular and spectroscopic analyses. Moreover, although metagenomic analysis revealed novel ssDNA sequences within the same cultures, direct evidence linking these sequences functionally or causally to NSE is still lacking.\u003c/p\u003e\n\u003cp\u003eAdditionally, our microscopic characterization was inherently limited by the resolution constraints of optical microscopy, restricting detailed ultrastructural interpretation. The scarcity and low density of NSE within cultures further constrained the scope and depth of analysis, highlighting the need for improved enrichment and isolation methods. Despite rigorous controls to minimize contamination risks, we cannot fully exclude the possibility of an unidentified environmental origin of these structures. Future studies should include independent validation through complementary analytical techniques, enrichment procedures, and rigorous replication to clarify these uncertainties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003ethe author declares no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e the author would like to thank Carlos Vello Costal and his team at for their invaluable assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdamala, K., \u0026amp; Szostak, J. (2013 ). Nonenzymatic template-directed RNA synthesis inside model protocells. Science, 342(6162), 1098-100. doi:10.1126/science.1241888.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlbers, S., \u0026amp; Jarrell, K. (2015 ). The archaellum: how Archaea swim. Front Microbiol., 6, 23. doi: 10.3389/fmicb.2015.00023.\u003c/li\u003e\n \u003cli\u003eAswani, K. K. (2016 ). Emerging LED Technologies for Fluorescence Microscopy. Microscopy Today, 24 (4), 22 - 27.\u003c/li\u003e\n \u003cli\u003eBarber, D., \u0026amp; Scott, E. (2002 ). Origin of supposedly biogenic magnetite in the Martian meteorite Allan Hills 84001. Proc Natl Acad Sci U S A., 99(10), 6556-61. doi:10.1073/pnas.102045799.\u003c/li\u003e\n \u003cli\u003eBarber-Zucker, S., Keren-Khadmy, N., \u0026amp; Zarivach, R. (2016 ). From invagination to navigation: The story of magnetosome-associated proteins in magnetotactic bacteria. Protein Sci., 25(2), 338-51. doi:10.1002/pro.2827.\u003c/li\u003e\n \u003cli\u003eBazylinski, D., \u0026amp; Frankel, R. (2004). Magnetosome formation in prokaryotes. Nat Rev Microbiol., 2(3), 217-30. doi:10.1038/nrmicro842.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eBerg, H. (2018). Random Walks in Biology: New and Expanded Edition. Kindle Edition. Princeton, Nueva Jersey.: Princeton University Press (November 20, 2018).\u003c/li\u003e\n \u003cli\u003eBlakemore, R. (1975). Magnetotactic bacteria. Science, 190(4212), 377-9. doi:10.1126/science.170679\u003c/li\u003e\n \u003cli\u003eBouteiller, M., Dupont, C., Bourigault, Y., Latour, X., Barbey, C., Konto-Ghiorghi, Y., \u0026amp; Merieau, A. (2021 ). Pseudomonas Flagella: Generalities and Specificities. Int J Mol Sci. , 22(7), 3337. doi:10.3390/ijms22073337.\u003c/li\u003e\n \u003cli\u003eCao, P., Dey, A., Vassallo, C., \u0026amp; Wall, D. (2015). How Myxobacteria Cooperate. J Mol Biol., 427(23), 3709-21. doi:10.1016/j.jmb.2015.07.022.\u003c/li\u003e\n \u003cli\u003eCavicchioli, R., Siddiqui, K., Andrews, D., \u0026amp; Sowers, K. (2002 ). Low-temperature extremophiles and their applications. . Curr Opin Biotechnol., 13(3), 253-61. doi:10.1016/s0958-1669(02)00317-8.\u003c/li\u003e\n \u003cli\u003eChandra, G., \u0026amp; Chater, K. (2014 ). Developmental biology of Streptomyces from the perspective of 100 actinobacterial genome sequences. FEMS Microbiol Rev., 38(3), 345-79. doi:10.1111/1574-6976.12047.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChaudhuri, S., Lack, J., \u0026amp; Coates, J. (2001). Biogenic Magnetite Formation through Anaerobic Biooxidation of Fe(II). Appl Environ Microbiol, 67(6), 2844 - 2848. doi:https://doi.org/10.1128/AEM.67.6.2844-2848.2001\u003c/li\u003e\n \u003cli\u003eChen, I., \u0026amp; Walde, P. (2010). From self-assembled vesicles to protocells. . Cold Spring Harb Perspect Biol., 2(7), a002170. doi:10.1101/cshperspect.a002170.\u003c/li\u003e\n \u003cli\u003eChicco, A., \u0026amp; Sparagna, G. (2007). Role of cardiolipin alterations in mitochondrial dysfunction and disease. 292(1), C33-44. doi:10.1152/ajpcell.00243.2006.\u003c/li\u003e\n \u003cli\u003eChyba, C., \u0026amp; Hand, K. (2005). Astrobiology: The Study of the Living Universe. Annual Review of Astronomy and Astrophysics, 43, 31-74. doi:https://doi.org/10.1146/annurev.astro.43.051804.102202\u003c/li\u003e\n \u003cli\u003eC\u0026iacute;ft\u0026ccedil;\u0026iacute;oglu, N., Miller-Hjelle, M., Hjelle, J., \u0026amp; Kajander, E. (2002). Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method. Antimicrob Agents Chemother. , 46(7), 2077-86. doi:10.1128/AAC.46.7.2077-2086.2002.\u003c/li\u003e\n \u003cli\u003eCockell, C. (2014). Habitable worlds with no signs of life. Philos Trans A Math Phys Eng Sci., 372(2014), 20130082. doi:10.1098/rsta.2013.0082.\u003c/li\u003e\n \u003cli\u003eCoenye, T., \u0026amp; Vandamme, P. (2003). Diversity and significance of Burkholderia species occupying diverse ecological niches. Environ Microbiol., 5(9), 719-29. doi:10.1046/j.1462-2920.2003.00471.x.\u003c/li\u003e\n \u003cli\u003ede la Escosura, A. (2019). The Informational Substrate of Chemical Evolution: Implications for Abiogenesis. Life (Basel)., 9(3), v. doi:10.3390/life9030066.\u003c/li\u003e\n \u003cli\u003ede la Higuera, I., \u0026amp; L\u0026aacute;zaro, E. (2022). Viruses in astrobiology. Front Microbiol., 13, 1032918. doi:10.3389/fmicb.2022.1032918.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDelwart, E., \u0026amp; Li, L. (2012). Rapidly expanding genetic diversity and host range of the Circoviridae viral family and other Rep encoding small circular ssDNA genomes. Virus Res., 164((1-2)), 114-21. doi:10.1016/j.virusres.2011.11.021.\u003c/li\u003e\n \u003cli\u003eDesfarges, S., \u0026amp; Ciuffi, A. (2012). Viral Integration and Consequences on Host Gene Expression. Viruses: Essential Agents of Life., 25, 147\u0026ndash;75. doi:10.1007/978-94-007-4899-6_7.\u003c/li\u003e\n \u003cli\u003eDrexler, H., \u0026amp; Uphoff, C. (2002). Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology, 39(2), 75-90. doi:10.1023/A:1022913015916.\u003c/li\u003e\n \u003cli\u003eDrobot, B., Iglesias-Artola, J., Le Vay, K., Mayr, V., Kar, M., Kreysing, M., . . . Tang, T. (2018 ). Compartmentalised RNA catalysis in membrane-free coacervate protocells. Nat Commun. , 9(1), 3643. doi:10.1038/s41467-018-06072-w.\u003c/li\u003e\n \u003cli\u003eDusenbery, D. (1997). Minimum size limit for useful locomotion by free-swimming microbes. Proc Natl Acad Sci U S A. , 94(20), 10949-54. . doi:v\u003c/li\u003e\n \u003cli\u003eErrington, J., \u0026amp; Aart, L. (2020). Microbe Profile: Bacillus subtilis: model organism for cellular development, and industrial workhorse. Microbiology (Reading). , 166(5), 425-427. doi:10.1099/mic.0.000922.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFerus, M., Pietrucci, F., Saitta, A., Kn\u0026iacute;žek, A., Kubel\u0026iacute;k, P., Ivanek, O., . . . Civi\u0026scaron;, S. (2017 ). Formation of nucleobases in a Miller-Urey reducing atmosphere. Proc Natl Acad Sci U S A., 114(17), 4306-4311. doi:10.1073/pnas.1700010114.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFrankel, R., Bazylinski, D., Johnson, M., \u0026amp; Taylor, B. (1997 ). Magneto-aerotaxis in marine coccoid bacteria. Biophys J. , 73(2), 994-1000. doi:10.1016/S0006-3495(97)78132-3.\u003c/li\u003e\n \u003cli\u003eGarcia, J., Patel, B., \u0026amp; Ollivier, B. (2000). Taxonomic, phylogenetic, and ecological diversity of methanogenic Archaea. Anaerobe, 6(4), 205-26. doi:10.1006/anae.2000.0345.\u003c/li\u003e\n \u003cli\u003eGil, J., Mesa, V., Estrada-Ortiz, N., Lopez-Obando, M., G\u0026oacute;mez, A., \u0026amp; Pl\u0026aacute;cido, J. (2021). Viruses in Extreme Environments, Current Overview, and Biotechnological Potential. Viruses, 13(1), 81. doi:10.3390/v13010081.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGiovannoni, S., Tripp, H., Givan, S., Podar, M., Vergin, K., Baptista, D., . . . Mathur, E. (2005). Genome streamlining in a cosmopolitan oceanic bacterium. Science, 309(5738), 1242-5. doi:10.1126/science.1114057.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGlavin, D., Dworkin, J., Alexander, C., JC, A., \u0026amp; al.., e. (2025). Abundant ammonia and nitrogen-rich soluble organic matter in samples from asteroid (101955) Bennu. Nat Astron. doi:https://doi.org/10.1038/s41550-024-02472-9\u003c/li\u003e\n \u003cli\u003eG\u0026ouml;kerk\u0026uuml;\u0026ccedil;\u0026uuml;k, E., Tramier, M., \u0026amp; Bertolin, G. (2020). Imaging Mitochondrial Functions: from Fluorescent Dyes to Genetically-Encoded Sensors. Genes (Basel)., 11(2), 125. doi:10.3390/genes11020125.\u003c/li\u003e\n \u003cli\u003eHorneck, G., St\u0026ouml;ffler, D., Ott, S., Hornemann, U., Cockell, C., Moeller, R., . . . Artemieva, N. (2008). Microbial rock inhabitants survive hypervelocity impacts on Mars-like host planets: first phase of lithopanspermia experimentally tested. Astrobiology, 8(1), 17-44. doi:10.1089/ast.2007.0134.\u003c/li\u003e\n \u003cli\u003eHou, X., Zaks, T., Langer, R., \u0026amp; Dong, Y. (2021). Lipid nanoparticles for mRNA delivery. Nat Rev Mater., 6(12), 1078-1094. doi:10.1038/s41578-021-00358-0.\u003c/li\u003e\n \u003cli\u003eJarrell, K., \u0026amp; McBride, M. (2008). The surprisingly diverse ways that prokaryotes move. Nat Rev Microbiol., 6(6), 466-76. doi:10.1038/nrmicro1900.\u003c/li\u003e\n \u003cli\u003eJarrell, K., Ding, Y., Nair, D., \u0026amp; Siu, S. (2013 ). Surface appendages of archaea: structure, function, genetics and assembly. Life (Basel)., 3(1), 86-117. doi:10.3390/life3010086.\u003c/li\u003e\n \u003cli\u003eJia, T., Chandru, K., Hongo, Y., Afrin, R., Usui, T., Myojo, K., \u0026amp; Cleaves, H. 2. (2019). Membraneless polyester microdroplets as primordial compartments at the origins of life. Proc Natl Acad Sci U S A., 116(32), 15830-15835. doi:10.1073/pnas.1902336116.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJohnson, L., Walsh, M., Bockus, B., \u0026amp; Chen, L. (1981). Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. . J Cell Biol., 88(3), 526-35. doi:10.1083/jcb.88.3.526.\u003c/li\u003e\n \u003cli\u003eJordan, S., Rammu, H., Zheludev, I., Hartley, A., Mar\u0026eacute;chal, A., \u0026amp; Lane, N. (2019 ). Promotion of protocell self-assembly from mixed amphiphiles at the origin of life. Nat Ecol Evol., 3(12), 1705-1714. doi:10.1038/s41559-019-1015-y.\u003c/li\u003e\n \u003cli\u003eKajander, E., \u0026amp; Cift\u0026ccedil;ioglu, N. (1998). Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci U S A., 95(14), 8274-9. doi:10.1073/pnas.95.14.8274.\u003c/li\u003e\n \u003cli\u003eKasai, T., Nakane, D., Ishida, H., Ando, H., Kiso, M., \u0026amp; Miyata, M. (2013 ). Role of binding in Mycoplasma mobile and Mycoplasma pneumoniae gliding analyzed through inhibition by synthesized sialylated compounds. . J Bacteriol. , 195(3), 429-35. doi:10.1128/JB.01141-12.\u003c/li\u003e\n \u003cli\u003eKazlauskas, D., Varsani, A., Koonin, E., \u0026amp; Krupovic, M. (2019). Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids. Nat Commun., 10(1), 3425. doi:10.1038/s41467-019-11433-0.\u003c/li\u003e\n \u003cli\u003eKhan, S. (1997 ). Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev., 61(4), 442-55. doi:10.1128/mmbr.61.4.442-455.1997.\u003c/li\u003e\n \u003cli\u003eKlein, H. (2020). Stressed DNA replication generates stressed DNA. Proc Natl Acad Sci U S A., 117(19), 10108-10110. doi:10.1073/pnas.2005160117.\u003c/li\u003e\n \u003cli\u003eKoch, A. (1997). Microbial physiology and ecology of slow growth. Microbiol Mol Biol Rev., 61(3), 305-18. doi:10.1128/mmbr.61.3.305-318.1997.\u003c/li\u003e\n \u003cli\u003eKoga, S., Williams, D., Perriman, A., \u0026amp; Mann, S. (2011 ). Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model. . Nat Chem., 3(9), 720-4. doi:10.1038/nchem.1110.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKrupovic, M., Dolja, V., \u0026amp; Kooni, n. E. (2019). Origin of viruses: primordial replicators recruiting capsids from hosts. Nat Rev Microbiol., 17(7), 449-458. doi:10.1038/s41579-019-0205-6.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLabont\u0026eacute;, J., \u0026amp; Suttle, C. (2015). Corrigendum: Metagenomic and whole-genome analysis reveals new lineages of gokushoviruses and biogeographic separation in the sea. Front Microbiol. , 6, 114. doi:10.3389/fmicb.2015.00114.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLauga, E., \u0026amp; Powers, T. (2009). The hydrodynamics of swimming microorganisms. Reports on Progress in Physics, 72(9), 096601. doi:https://doi.org/10.1088/0034-4885/72/9/096601\u003c/li\u003e\n \u003cli\u003eLauro, F., McDougald, D., Thomas, T., Williams, T., Egan, S., Rice, S., . . . Cavicchioli, R. (2009 ). The genomic basis of trophic strategy in marine bacteria. Proc Natl Acad Sci U S A., 106(37), 15527-33. doi:10.1073/pnas.0903507106.\u003c/li\u003e\n \u003cli\u003eLiu, Y., \u0026amp; Whitman, W. (2008). Metabolic, phylogenetic, and ecological diversity of the methanogenic archaea. Ann N Y Acad Sci. , 1125, 171-89. doi:10.1196/annals.1419.019.\u003c/li\u003e\n \u003cli\u003eL\u0026oacute;pez Ram\u0026oacute;n y Cajal, C. (2025). Discovery of single-stranded DNA in meteorite-derived cultures: evidence of novel genetic elements. bioRxiv, 633545. doi:https://doi.org/10.1101/2025.01.20.633545\u003c/li\u003e\n \u003cli\u003eMateescu, B., Kowal, E., van, B. B., Bartel, S., Bhattacharyya, S., Buz\u0026aacute;s, E., . . . Lunav. (2017). Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper. J Extracell Vesicles., 6(1), 1286095. doi:10.1080/20013078.2017.1286095.\u003c/li\u003e\n \u003cli\u003eMaurer, S., Deamer, D., Boncella, J., \u0026amp; Monnard, P. (2009). Chemical evolution of amphiphiles: glycerol monoacyl derivatives stabilize plausible prebiotic membranes. Astrobiology, 9(10), 979-87. doi:10.1089/ast.2009.0384.\u003c/li\u003e\n \u003cli\u003eMcCoy, T., Russell, S., Zega, T., Thomas-Keprta, K., Singerling, S., Brenker, F., . . . Jones, R. (2025). An evaporite sequence from ancient brine recorded in Bennu samples. . Nature, 637((8048)), 1072-1077. doi:10.1038/s41586-024-08495-6.\u003c/li\u003e\n \u003cli\u003eMcKenney, P., Driks, A., \u0026amp; Eichenberger, P. (2013). The Bacillus subtilis endospore: assembly and functions of the multilayered coat. . Nat Rev Microbiol, 11(1), 33\u0026ndash;44. doi:10.1038/nrmicro2921.\u003c/li\u003e\n \u003cli\u003eMolecular Probes, I. (Revised June 25, 2008). MitoTracker\u0026reg; Mitochondrion-Selective Probes. Invitrogen Corporation.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMukherjee, S., \u0026amp; Kearns, D. (2014). The structure and regulation of flagella in Bacillus subtilis. Annu Rev Genet., 48, 319-40. doi:10.1146/annurev-genet-120213-092406.\u003c/li\u003e\n \u003cli\u003eNewton, R., Griffin, L., Bowles, K., Meile, C., Gifford, S., Givens, C., . . . Moran, M. (2010). Genome characteristics of a generalist marine bacterial lineage. ISME J., 4(6), 784-98. doi:10.1038/ismej.2009.150.\u003c/li\u003e\n \u003cli\u003eNicholson, W., Munakata, N., Horneck, G., Melosh, H., \u0026amp; Setlow, P. (2000). Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev., 64(3), 548-72. doi:10.1128/MMBR.64.3.548-572.2000.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNikfarjam, L., \u0026amp; Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell J., 13(4), 203-12.\u003c/li\u003e\n \u003cli\u003eOren, A. (2002). Diversity of halophilic microorganisms: environments, phylogeny, physiology, and applications. J Ind Microbiol Biotechnol, 28(1), 56-63. doi:10.1038/sj/jim/7000176.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePal, A., \u0026amp; Levy, Y. (2019). Structure, stability and specificity of the binding of ssDNA and ssRNA with proteins. PLoS Comput Biol. , 15(4), e1006768. doi:10.1371/journal.pcbi.1006768.\u003c/li\u003e\n \u003cli\u003ePatel, B., Percivalle, C., Ritson, D., Duffy, C., \u0026amp; Sutherland, J. (2015). Common origins of RNA, protein and lipid precursors in a cyanosulfidic protometabolism. Nat Chem., 7(4), 301-7. doi:10.1038/nchem.2202.\u003c/li\u003e\n \u003cli\u003ePearce, B., Pudritz, R., Semenov, D., \u0026amp; Henning, T. (2017). Origin of the RNA world: The fate of nucleobases in warm little ponds. Proc Natl Acad Sci U S A., 114(43), 11327-11332. doi:10.1073/pnas.1710339114.\u003c/li\u003e\n \u003cli\u003ePendergrass, W., Wolf, N., \u0026amp; Poot, M. (2004). Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A., 61(2), 162-9. doi:10.1002/cyto.a.20033.\u003c/li\u003e\n \u003cli\u003ePham, V., \u0026amp; Kim, J. (2012). Cultivation of unculturable soil bacteria. Trends Biotechnol., 30(9), 475-84. doi:10.1016/j.tibtech.2012.05.007.\u003c/li\u003e\n \u003cli\u003ePietil\u0026auml;, M., Roine, E., Paulin, L., Kalkkinen, N., \u0026amp; Bamford, D. (2009). An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope. Mol Microbiol., 72(2), 307-19. doi:10.1111/j.1365-2958.2009.06642.x\u003c/li\u003e\n \u003cli\u003ePizzarello, S. (2006). The chemistry of life\u0026apos;s origin: a carbonaceous meteorite perspective. Acc Chem Res., 39(4), 231-7. doi:10.1021/ar050049f.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePodar, M., Makarova, K., Graham, D. W., Koonin, E., \u0026amp; Reysenbach, A. (2013). Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park. Biol Direct., 8, 9. doi:10.1186/1745-6150-8-9.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePresley, A., Fuller, K., \u0026amp; Arriaga, E. (2003). MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr B Analyt Technol Biomed Life Sci. , 793(1), 141-50. doi:doi: 10.1016/s1570-0232(03)00371-4.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRajamani, S., Vlassov, A., Benner, S., Coombs, A., Olasagasti, F., \u0026amp; Deamer, D. (2008). Lipid-assisted synthesis of RNA-like polymers from mononucleotides. Orig Life Evol Biosph., 38(1), 57-74. doi:10.1007/s11084-007-9113-2.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eRazin, S., Yogev, D., \u0026amp; Naot, Y. (1998). Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev., 62(4), 1094-156. doi:10.1128/MMBR.62.4.1094-1156.1998.\u003c/li\u003e\n \u003cli\u003eReufer, M. B., Schwarz-Linek, J., Martinez, V., Morozov, A., Arlt, J., Trubitsyn, D., . . . Poon, W. (2014). Switching of Swimming Modes in Magnetospirillium gryphiswaldense. Biophys J. , 106(1), 37-46. doi:10.1016/j.bpj.2013.10.038.\u003c/li\u003e\n \u003cli\u003eRothschild, L., \u0026amp; Mancinelli, R. (2001). Life in extreme environments. 409(6823), 1092-101. doi:10.1038/35059215.\u003c/li\u003e\n \u003cli\u003eSampedro, I., Parales, R., Krell, T., \u0026amp; Hill, J. (2015). Pseudomonas chemotaxis. FEMS Microbiol Rev., 39(1), 17-46. doi:10.1111/1574-6976.12081.\u003c/li\u003e\n \u003cli\u003eScaduto, R. J., \u0026amp; Grotyohann, L. (1999 ). Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives. . Biophys J. , 76((1 Pt 1)), 469-77. doi:10.1016/S0006-3495(99)77214-0.\u003c/li\u003e\n \u003cli\u003eSephton, M. (2002). Organic compounds in carbonaceous meteorites. Nat Prod Rep, 19(3), 292-311. doi:10.1039/b103775g.\u003c/li\u003e\n \u003cli\u003eSetlow, P. (2014). Spore Resistance Properties. Microbiol Spectr., 2(5), TBS-0003-2012. doi:10.1128/microbiolspec.TBS-0003-2012.\u003c/li\u003e\n \u003cli\u003eShatilovich, A., Gade, V., Pippel, M., Hoffmeyer, T., Tchesunov, A., Stevens, L., . . . Kurzchalia, T. (2023 ). A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva. PLoS Genet., 19(7), e1010798. doi:10.1371/journal.pgen.1010798.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSilby, M., Winstanley, C., Godfrey, S., Lev, y. S., \u0026amp; Jackson, R. (2011). Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev., 35(4), 652-80. doi:10.1111/j.1574-6976.2011.00269.x.\u003c/li\u003e\n \u003cli\u003eSutherland, J. (2017). Opinion: Studies on the origin of life \u0026mdash; the end of the beginning. Nat Rev Chem, 1, 0012 . doi:https://doi.org/10.1038/s41570-016-0012\u003c/li\u003e\n \u003cli\u003eSzostak, J. (2012). The eightfold path to non-enzymatic RNA replication. J Syst Chem, 3, 2. doi:https://doi.org/10.1186/1759-2208-3-2\u003c/li\u003e\n \u003cli\u003eValentine, D. (2007). Adaptations to energy stress dictate the ecology and evolution of the Archaea. Nat Rev Microbiol., 5(4), 316-23. doi:10.1038/nrmicro1619.\u003c/li\u003e\n \u003cli\u003eVan Cappellen, P. (2003). Biomineralization and Global Biogeochemical Cycles. Reviews in Mineralogy and Geochemistry, 54(1), 357\u0026ndash;381. doi:doi: https://doi.org/10.2113/0540357\u003c/li\u003e\n \u003cli\u003eWaters, E., Hohn, M., Ahel, I., Graham, D., Adams, M., Barnstead, M., . . . Noordewier, M. (2003). The genome of Nanoarchaeum equitans: insights into early archaeal evolution and derived parasitism. Proc Natl Acad Sci U S A, 100(22), 12984-8. doi:10.1073/pnas.1735403100.\u003c/li\u003e\n \u003cli\u003eWu, C., Martel, J., Wong, T., Young, D., Liu, C., Lin, C., \u0026amp; Young, J. (2016). Formation and characteristics of biomimetic mineralo-organic particles in natural surface water. Sci Rep., 6, 28817. doi:10.1038/srep28817\u003c/li\u003e\n \u003cli\u003eXiao, B., Deng, X., Zhou, W., \u0026amp; Tan, E. (2016 ). Flow Cytometry-Based Assessment of Mitophagy Using MitoTracker. Front Cell Neurosci., 10, 76. doi:10.3389/fncel.2016.00076.\u003c/li\u003e\n \u003cli\u003eZhang, M., Mileykovskaya, E., \u0026amp; Dowhan, W. (2005). Cardiolipin is essential for organization of complexes III and IV into a supercomplex in intact yeast mitochondria. J Biol Chem., 280(33), 29403-8. doi:10.1074/jbc.M504955200.\u003c/li\u003e\n \u003cli\u003eZhang, W., \u0026amp; Wu, L. (2020). Flagella and Swimming Behavior of Marine Magnetotactic Bacteria. Biomolecules, 10(3), 460. doi:10.3390/biom10030460.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Alvaro Cunqueiro Hospital, University Hospital Complex of Vigo, Spain.","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Novel Structured Entities (NSE), Meteorite Cultures, Live Optical LED (LOL) Microscopy, Astrobiology, Microscopy, ssDNA Persistence, Prebiotic Evolution","lastPublishedDoi":"10.21203/rs.3.rs-6185543/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6185543/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe discovery of Novel Structured Entities (NSE) in meteorite-derived cultures provides an opportunity to investigate structured formations with unique physicochemical properties and their potential role in the stabilization of genetic elements. This study employs Live Optical LED microscopy to characterize and dynamically visualize the structural properties and organizational stages of NSE under controlled conditions. Meteorite fragments from diverse locations and compositions were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium or sterile distilled water, enabling the observation of NSE morphologies, including motile vesicular structures (Free Forms), protective biofilms (Biofilm Forms), and highly mineralized laminated fibers (Resistant Forms). Staining with MTG revealed fluorescence across all observed NSE stages, suggesting an interaction with specific structural components rather than direct metabolic activity.\u003c/p\u003e \u003cp\u003eOur findings indicate that these structured formations emerge in meteorite-derived cultures and exhibit properties suggesting resilience under extreme environmental conditions. While their organized morphology and co-occurrence with ssDNA sequences are intriguing, further research is required to determine whether this association holds any functional significance or results from unrelated culture dynamics. These results provide a structural framework for a parallel genomic study, which detected previously uncharacterized single-stranded DNA (ssDNA) sequences within the same cultures, warranting further investigation into potential associations.\u003c/p\u003e \u003cp\u003eWhile the origins and nature of these structured formations remain to be fully elucidated, these findings underscore the importance of integrating molecular, structural, and functional analyses to explore the persistence of genetic elements in extreme environments and their potential implications for prebiotic evolution.\u003c/p\u003e","manuscriptTitle":"Microscopic Characterization and Dynamic Visualization of Novel Structured Entities in Meteorite Cultures.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 08:27:50","doi":"10.21203/rs.3.rs-6185543/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e0c79c7a-95cd-4a79-8b43-3b586e2eaa14","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45403703,"name":"Astrobiology"},{"id":45403704,"name":"Evolutionary Biology"},{"id":45403705,"name":"Meteoritics"},{"id":45403706,"name":"Astrochemistry"}],"tags":[],"updatedAt":"2025-03-12T08:27:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 08:27:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6185543","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6185543","identity":"rs-6185543","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}