{"paper_id":"05adcf60-d120-47e0-ae3a-d5b0ee59f692","body_text":"Shotgun proteomic analysis of the caterpillar Lonomia obliqua (Lepitoptera, Saturniidae) hemolymph and effects in rat hippocampal neurons culture | 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 Shotgun proteomic analysis of the caterpillar Lonomia obliqua (Lepitoptera, Saturniidae) hemolymph and effects in rat hippocampal neurons culture Silviane Maggi, Antonio Frederico Michel Pinto, Mariana Sayuri Berto Udo, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5291061/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 Study of substances with potentially neuroprotective has been one of the research focus on drugs development. Toxic proteins of Lonomia obliqua caterpillars, which have caused several accidents in southern Brazil, were identified in the hemolymph with anti-apoptotic activity. This study aims the evaluation of the protein profile and the hemolymph effect on cell viability of rats’ primary cultured hippocampal neurons after apoptosis induction. Semi-quantitative shotgun proteomics approach was used to evaluate the protein profile of 3 caterpillars lots of different origin. Were identified a total of 76 proteins, 71 in hemolymph and 40 in fractions. Antiviral protein predominated in crude hemolymph, following by serine proteases, hemolins and protease inhibitors. In fractions were identified hemolins, serine proteases and protease inhibitors. The treatment of rats’ primary cultured hippocampal neurons with the chromatographic fraction at concentration of 0.05 and 0.10% (v/v) for 24 hours, with subsequently apoptosis induction was able to maintain cell viability significantly higher than positive control. Hemolymph protein composition can show qualitative and quantitative variations intra species when compared different origins animals and consequently exposed to various environmental factors. The results shown on this study may contribute to the identification of proteins with potential use as neuroprotective in degenerative conditions. Lonomia shotgun proteomic apoptosis hippocampal culture Figures Figure 1 Figure 2 Figure 3 1. Introduction Insects represent about 75% of all invertebrate animals, are among the more adapted to life on earth. These organisms are capable of producing a number of chemicals that help them survive the environmental attacks during their evolution (Ratcliffe et al ., 2011). Thus, evolutionarily, poisons and venoms animals were developed for the purpose of defense against predators or prey capture, causing physiological changes in natural enemies. However, they have potential application as new therapeutic drugs and have been the subject of research (Calvete, 2009). Several accidents involving the larval form of the Lonomia obliqua Walker, 1855 (Lepidoptera, Saturniidae) have been reported in southern Brazil, since the 80s (Duarte et al. , 1990; Duarte et al. , 1994; Kelen et al. , 1995; Duarte et al. , 1996; Zannin et al. , 2003). Individuals who had contact with bristles of L. obliqua caterpillars can manifest a hemorrhagic syndrome associated with consumption coagulopathy which may include intravascular hemolysis and acute renal failure. Individual characteristics of the victims, intensity of exposure to poison and the number of animals involved, may determine slight, serious or even fatal accidents (Duarte et al ., 1996; Fan et al ., 1998; Gamborgi et al ., 2006; Malaque et al ., 2006; Riella et al ., 2008; Basulado et al ., 2008). Physiological activity presented by the venom in their victims initiated several studies on its composition, in order to develop therapy for poisoning and identify potential resources for the treatment of various conditions. Among the compounds identified a protein has been well characterized, called Lopap ( Lonomia Prothrombin Activator Protease), but the literature still lacks studies to elucidate the overall protein content of venom composition and biological fluids caterpillar, which yet remains incomplete. Proteomes are highly dynamic and may change with development and insect environment (Wilkins et al ., 1997). Among the wide possibilities of bioactive molecules that insects can provide protein compounds with anti-apoptotic activity have been described (Rhee et al. , 2013; Choi et al. , 2002; Kim et al ., 2001; Rhee and Park, 2000). This activity may be interesting in pathologies that develop with cell loss by apoptosis, such as neurodegenerative. L. obliqua hemolymph anti-apoptotic activity has been described for insect cells and mammals forward physical inducers, biological and chemical (Vieira et al ., 2010; Mendonça et al ., 2008; Souza et al ., 2005; Maranga et al ., 2003), but have not yet been described studies conducted with nerve cells. This study aimed identify hemolymph and fractions protein composition of three caterpillar colonies from different cities, collected at different times and feeding on various plants, and evaluate if there were significant changes between profiles of the same. Still, the effect of L. obliqua caterpillars crude hemolymph and their chromatographic fractions was evaluated on cell viability of rat primary hippocampal cell cultures. 2. Materials and Methods Hemolymph collection. Lonomia obliqua caterpillars was collected in state of Rio Grande do Sul (South of Brazil) and frozen at -20 °C by 4 h. Hemolymph was harvested from sixty instar larvae after cutting bristles. The collected hemolymph was centrifuged by 14,000 rpm for 20 min, the supernatant was filtered through a 0.22 µm membrane filter and stored at -20 °C. Hemolymph Semi-Purified Fraction. Semi-purified fraction with anti-apoptotic activity was obtained from Butantan Institute (São Paulo, Brazil) (Souza et al. , 2005). Briefly 0.5 mL of total hemolymph was fractionated on an AKTA Purifier chromatography system equipped with a Resource Q ion exchange column (Amersham Pharmacia Biotech, USA) at a rate of 0.5 mL min -1 and eluted at a linear gradient (0-100%), TrisHCl 20 mmol L -1 /Tris HCl – NaCl 1 mol L -1 , pH 8.0. The eluate was monitored at 280, 254 and 214 nm and harvested in fractions of 1 mL. Animals. Wistar rats male (weighing 200 g, 100 days old) and virgin female (weighing 190 g, 90 days old) were obtained from School of Pharmaceutical Sciences/Chemistry Institute, University of São Paulo, São Paulo, Brazil. Females were caged overnight with males and copulation was verified in the morning by detection of a vaginal plug. Pregnant females were housed under controlled temperature (22±1 °C) on a 12:12 hours light/dark cycle. Food and water were provided ad libitum . Fetuses were obtained at 18-19 days of pregnancy (E18-E-19). Pregnant rats were anesthetized with pentobarbital (45 mg Kg -1 ) and the fetuses were rapidly euthanized by decapitation to remove their hippocampi. All experiments were conducted according to the guidelines issued by the National Council for Animal Experimentation and this study was approved by the Animal Uses Ethic Committee of the Pharmaceutical Sciences School of University of São Paulo. Cell culture. Rat embryonic hippocampal primary cultures were obtained according Garcia et al . (2012) with slightly modifications. Primary cultures were obtained by hippocampal neurons dissociation from hippocampi of E18-E19 Wistar rat embryos. Hippocampi were maintained in solution containing cooled neurobasal medium (Gibco, USA) with 100 U mL -1 penicillin and 100 µg mL -1 streptomycin. Tissues were washed with Hank’s Balanced Salt Solution (HBSS; Gibco, USA) and fragmented mechanically. Cell isolation was obtained by proteolytic digestion with trypsin according Jahr and Stevens (1987) and Silva et al. (2006). Hippocampi fragments were incubated with trypsin 0,25% (Gibco, USA), pH 7.2-7.4, at 37 °C for 10 min. Reaction was stopped with HBSS containing 277.5 U mL -1 DNAse (Sigma, USA) and 10% fetal bovine serum (Gibco, USA), pH 7.2-7.4. Cells were then dispersed mechanically with Pasteur pipettes of different diameters. After, hippocampal cells were resuspended in neurobasal medium (Gibco, USA) supplemented with 0.5 mmol L -1 L-glutamine (Gibco, USA), 25 µmol L -1 L-glutamic acid (Sigma, USA), 100 U mL -1 penicillin: 100 µg mL -1 streptomycin (Sigma, USA) and 2% B27 supplement (Gibco, USA), to reduce glial cell proliferation (Brewer et al ., 1993; Silva et al ., 2006). Cells suspension was then plated onto 0.01% poly-L-lysine-coated (Sigma, USA) 96-well culture plates at a density of 5.10 4 cells per well and incubated for 7-8 days at 37°C, in humidified atmosphere of 5% CO 2 for hippocampal neurons maturation. Half of the medium culture was replaced for the same volume of fresh medium with the same composition each 48 h. On 6-8 day, cultured cells were incubated with the treatments according to the experiment. Previous study by immunohistochemistry (Garcia et al. , 2012) showed a predominance of neurons in this culture, with 92% of neurons and 8% of astrocytes. Apoptosis induction. Hippocampal cells (5.10 4 cells/well) were treated with 10 and 30 µmol L -1 H 2 O 2 solutions [from H 2 O 2 stock solution 30% (Merck, Germany), into supplemented neurobasal medium] fresh made, for 30 min (37°C, 5% CO 2 ), after treatment with hemolymph and fractions in different concentrations. Exposure to H 2 O 2 solutions was performed within 5 minutes of dilution, in the dark. Cell viability. Cells viability was evaluated by MTT-reduction assay (Mosman, 1983; Liu et al., 1997). Active metabolically cells are capable to cleave 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by reduction of this yellow salt to formazan crystals, a purple compound, by mitochondrial reductases enzymes of viable cells. Formazan crystals can be measured by absorbance at 570 nm and directly correlated with viable cells number. For the assessment of cell viability, primary hipocampal cells in culture (5.10 4 cells/well; 7-8° culture day) were incubated with hemolymph or fraction at 0.05 and 0.1% (into supplemented neurobasal medium) for 1 and 24 h (37°C, 5% CO 2 ). After the period of incubation, cells were treated with 10 and 30 µmol L -1 H 2 O 2 solutions (into supplemented neurobasal medium) and incubated for 30 min (37°C, 5% CO 2 ). Treatments were then replaced by 100 µL 0.5 mg mL -1 MTT (Sigma-Aldrich Co., USA) solution and plates were incubated for 3 h (37°C, 5% CO 2 ). Then, MTT solution was removed and 200 µL of dimethyl sulfoxide (DMSO, Synth, Brazil) was added to each well. After, plates were shaking for 30 min and the absorbance was measured at 570 nm in a multiwell plate reader Synergy H1 Hybrid Reader (Biotek Instruments Inc., USA). Statistical analysis. Data were reported as means ± SD (standard deviation) and statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test ( p < 0.05 was accepted as statistically significant). Experiments were conducted in quadruplicate from at least three independent experiments. Assays were analyzed by Prism 5 software (Graph Pad Software, USA). 2.1 Shotgun analysis Sample preparation. Protein concentration of the samples was determined using a Bradford assay (Bradford, 1976) with bovine serum albumin (BSA) as a standard. Proteins were concentrated according Wessel and Flügge (1984). Briefly, 20 µg (total proteins) of each sample were submitted to protein precipitation with chloroform and methanol (1:3:1:3, sample:methanol:chloroform:destilated water) followed by centrifugation at 17,000 rpm, 10 min. Supernatant was removed and were added three volumes of methanol (centrifugation at 17,000 rpm, 10 min). After, liquid phase was discarded and precipitated was maintained at room temperature to dry. Precipitates of concentrated proteins were resuspended in buffer urea (8 mol L -1 ), tris (0.1 mol L -1 ) and was followed the digestion protocol adapted of Klammer and MacCoss (2006). Briefly, disulfide bonds were reduced by DTT 10 mmol L -1 addition (37ºC, 20 min) followed alkylation by IAA 50 mmol L -1 addition (room temperature, in the dark, 20 min). After, urea was diluted to 2 mol L -1 by 60 µL of tris 0.1 mol L -1 addition. Then, proteins were digested with trypsin (1:50, enzyme:substrate) in presence of CaCl 2 1 mmol L -1 (37°C, 18 h). Reaction was stopped by formic acid addition (5% v/v, final concentration). NanoLC LTQ-XL Orbitrap MS/MS. Chromatography separations of tryptic peptide mixture was obtained by nanoLC Ultra (nanoLC Ultra 1D plus, Eksigent, USA) equipped with autosampler nanoLC AS-2 (Eksigent, USA) and connected to LTQ-XL Orbitrap Discovery MS/MS (Thermo Fischer Scientific, USA), containing a nano- electrospray ionization source (Thermo Fischer Scientific, USA). Analytical capillary columns (100 µm × 20 cm) and pre-columns (150 µm × 2 cm) was packaged in house with phase-reversed C18 (5 µm ODS-AQ C18, Yamamura Chemical Lab). Hemolymph and chromatography fractions was loaded in autosampler with injection volume 10 µL and at a flow rate of 1 µL mL -1 for 15 min. Step gradient mobile phase A (5% acetonitrile, 0.1% formic acid in water) was used to chromatography separations of 120 min (fractions) or 360 min (hemolymph) to mobile phase B (90% acetonitrile, 0.1% formic acid): 0-5% B in 5 min; 5-25% B in 60 min; 25-50% B in 20 min; 50-80% B in 15 min; 80% isocratic B for 5 min; 80-5% B in 1 min; 5% isocratic B for 14 min at a flow rate of 400 nL min -1 ). Positive ion mode was employed and normalized collision energy was set to 35%. Full scan MS spectra were acquired from m/z 400-1,600 at a resolving power of 30,000, followed by acquisition of 8 MS2 mode spectra of the most abundant ions. Fragmentation was obtained by dissociated induced collision (CID), with Q activation = 0.250, time of activation = 30 ms and isolation amplitude = 1 Da. Acquired peptide masses were added to dynamic exclusion list with size of 100 ions and time of permanence of 30 s during acquisition of MS2 spectra. Spray voltage used were 2.2 kV, capillary temperature of 275°C, capillary voltage of 34 V, and the collision gas used was helium. Data analysis. For protein identification, a no-redundant sequences data bank referred to entries of individuals proteins for “ Lonomia ” presents in NCBI data bank (http://www.ncbi.nlm.nih.gov) was constructed. From this bank, RAW files of MS-2 spectra was searched using Comet search software (Eng et al. , 2013). Identification proteins and peptides was obtained using Pattern Lab for Proteomics software from homology with data bank sequences (Carvalho et al ., 2012). Candidates peptides were considered those containing one or two tryptic ends. Cysteine carbamidomethylation was adopted as fixed modification. Tolerance of 50 ppm to precursors ions and 1 Da to fragment ions were employed on the data search. Search Engine Processor software (Carvalho et al., 2012) was used to filter identified spectra. Parameters Xcorr , DeltaCN , DeltaMass , Peaks Matched e Spec Count Score were used to generate a Bayesian score. Cutoff was established to accept 1% false-positive, based on reversed data base identifications. Furthermore, 6 residues were adopted as minimum sequence length. Results were post processed to set identifications less than 10 ppm of mass variation. 3. Results and discussion 3.1 Hemolymph and chromatographic fraction protein identification by NanoLC MS/MS Studies were conducted using a gel-free proteomic strategic in a nanoLC/LTQ- Orbitrap system. A total of 71 distinct hemolymph proteins were identified from 3 lots of caterpillars, with 34 common proteins between the same. In fractions was found 40 total distinct proteins, with 9 shared between the 3 lots. A Venn diagram displays the results in Fig. 1 . Fig. 1 - Venn diagram depicting the total number of proteins identified in 3 lots of hemolymph (H) and chromatographic fractions (CF) as well as the number of overlapped proteins. Considering hemolymph and CF samples, 76 different proteins were identified, with molecular weights ranging from 2,107.1 to 71,540.4 Da: 71 in hemolymph and 40 in CF, with 34 common proteins between then. Five proteins were found only in CF and 36 just in hemolymph. Related to CF, a proportionately larger number of peptides was generated by hemolymph, due to the larger number of proteins present in this. The number of peptides produced by proteolytic digestion is proportional to abundance of its protein in a sample (Liu et al., 2004). Thus, peptides in larger quantity were selected to MS2 more frequently, generating a larger number of spectral counts which is proportional to the abundance of each protein in the sample. Five proteins were identified only in CF, since in the crude sample, more peptides can compete to the selection of fragmentation within the same cycle of acquisition, and thus, peptides signal with less intensity can be suppressed at the expense of others compromising their identification. Protein identification in L. obliqua samples by shotgun analysis is impaired by the limited range of data contained in the database for this specie. Proteins amino acids sequences data found in databases are mainly from transcriptomic studies (Veiga et al. , 2005). Serine proteases (45%), hemolins (38%), protease inhibitors (7%) and antiviral protein (5%) were found in CF, as well as sensory proteins, heat shock proteins, cysteine proteases and lectins less expressively. In hemolymph were found predominantly antiviral protein (24%), serine proteases (23%), hemolins (16%) and protease inhibitors (10%). Heat shock proteins, serpins, sensory proteins, oxidoreductases, lectins, lyases and ribonucleases also were identified. Other found proteins together represent 20% of the total, and are structural proteins or proteins involved in metabolic functions. Significant qualitative and quantitative variations in hemolymph amino acids concentration and protein composition were related to insect age and stage of development (Roman and Jegorov, 1991; Whitmore and Gilbert, 1974), diet (Ortel, 1995) and ambient temperature (Roman and Jegorov, 1991; Cui et al. , 2011). Thus, this can have determined differences in protein composition observed in the three lots of hemolymph. Table 1 - Identified proteins on hemolymph and chromatographic fractions samples by shotgun . Insects present a metamorphosis process where a combination of grow sinaling, activation, differentiation and specific tissues physiological apoptosis is highly regulated. Probably substances responsible for this signaling circulate in hemolymph, which makes their interesting for study to identification of bioactive compounds with biotechnological potential (Maranga et al. , 2003). 3.2 Hemolymph and CF effects on H 2 O 2 - treated cells 3.2.1 Hemolymph Mitochondrial metabolism of 0.05% and 0.1% hemolymph treated cells for 1 h and subsequently subjected to apoptosis induction with H 2 O 2 10 μmol L -1 was significantly reduced ( p < 0.01) compared to control without inducing apoptosis. However, when compared to apoptosis induction control with the same H 2 O 2 concentration, there was no significant difference. Viability of 0.05% and 0.1% hemolymph cells treated for 1 h with apoptosis induced by H 2 O 2 30 μmol L -1 also had a significant reduction ( p < 0.001) relative to untreated control, but there was no significant difference when the comparison was made with H 2 O 2 30 μmol L -1 control. Treatment with 0.05% hemolymph, H2O2 10 mol L -1 showed no statistical difference compared to control and H2O2 10 μmolL -1 . Cells submitted to 0.1% hemolymph, H2O2 10 μ mol L -1 had significant reduction ( p < 0.001) in cell viability compared to control and H2O2 10μmol L -1 ( p < 0.05). Cells with 0.05% and 0.1% hemolymph for 24 h, exposed to H 2 O 2 30mol L -1 had significantly reduction viability ( p < 0.001) relative to control and was not difference in comparison with H 2 O 2 30 μmol L -1 . Presence of other active substances that may play an inhibitory effect on cell viability and proliferation, such as A2 phospholipase involved in hemolytic activity presented by the poison and hyaluronidase activity should be considered in evaluating the crude hemolymph effect (Heinen et al ., 2014). Fig. 2 - Evaluation of cell viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the three independents assays); H (Hemolymph; concentrations at 0.05% or 0.1% v/v); H 2 O 2 (Positive control; concentrations at 10 or 30 µM). * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison). 3.2.2 Chromatographic Fraction (CF) Treated cells with 0.05% and 0.1% CF for 1 h and H 2 O 2 10mol L -1 produced viable cells significant reduction ( p < 0.001 and p < 0.01 respectively) compared to untreated control and 0.05% and 0.1% CF for 1 h, H 2 O 2 30 mol L -1 ( p < 0.001). There were no significant differences when comparing CF and H 2 O 2 10 mol L -1 either between CF and H 2 O 2 30mol L -1 . For 0.05% and 0.1% CF (24 h),H 2 O 2 10 mol L -1 there is not statistical difference compared to control, however occurred cell viability increase related to H 2 O 2 10 mol L -1 ( p < 0.05 and p < 0.01, respectively). Cells with 0.05%and 0.1% for24 h, H 2 O 2 30 mol L -1 had significant reduction viability ( p < 0.01) regarding control. Increase in viable cells ( p < 0.05) was produced by 0.05% CF, H2O2 30 μmol L-1 related to H2O2 30 μmol L-1 while 0.1% CF, H2O2 30 μmol L-1 had no difference. Fig. 3 - Evaluation of celL viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the five independents assays); CF (Chromatographic fraction; concentrations at 0.05% or 0.1% v/v); H 2 O 2 (Positive control; concentrations at 10 or 30 µM). * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison). After 24 h of exposure the rat hipocampal neurons culture to CF, and subsequently exposure to H 2 O 2 for 30 min, the cellular viability kept higher than positive control (only H 2 O 2 ), but a reduction in neuronal viability was observed related to control (without oxidative induction). Several studies have shown the property of substances from insects hemolymph in the inhibition of apoptosis promoted by different inducers (physical, chemical and biological), in insects and mammals cell cultures (Rhee et al. , 2013; Vieira et al. , 2010; Mendonça et al. , 2008; Souza et al. , 2005; Maranga et al. , 2003; Choi et al. , 2002; Kim et al. , 2001; Rhee and Park, 2000). The effect in a variety of cells opposite the apoptosis induction by multiple mechanisms suggests that it may act in some apoptosis conserved way (Heinen et al. , 2014). L. obliqua hemolymph anti-apoptotic activity was observed in Sf-9 insect cells (Souza et al. , 2005; Maranga et al ., 2003; Vieira et al. , 2010), in HEK-293 human cells (Mendonça et al ., 2007) and in mammalian cells V-79 (Heinen et al. , 2014). Mendonça et al . (2007) observed that exposure to hemolymph and chromatographic fractions produced high electrochemical potential in the mitochondrial membrane maintenance. Cytoprotective activity was described too in leucocytes and endothelial cells (Chudzinski-Tavassi et al., 2010), in HUVECs (Fritzen et al., 2005) and in fibroblasts FN-1 ( rLosac – recombinant Lonomia obliqua Stuart-Factor Activator) (Alvarez-Flores et al. , 2012), but associated to different proteins from L. obliqua . Further studies to verify expressed proteins from samples treated cultures may indicate if factors such as cells from animals tissues adaptation differences to in vitro condition can be related to results obtained. Moreover, it is still unknown anti-apoptotic protein interaction with specific cellular receptors that may not be present or not be available in quantities sufficient for the manifestation of the effects in the studied cell. The structure of the anti-apoptotic effects responsible protein remains without being elucidated and thus the prediction of their susceptibility to degradation or inactivation are also compromised. 4. Conclusion As demonstrated in this study, animal venoms protein composition such as L. obliqua can provide qualitative and quantitative variations intra species when Compared different origins animals and consequently exposed to various environmental factors. Concentration of free ions naturally present in the hemolymph can likewise differ between animals and produce shift in the elution of specific proteins during ion exchange chromatography making not reproducible elution pattern of the fractions. Thus, proteins present in fraction used in this study may differ over those described in the literature using the same fractionation strategy. Still, there may be synergistic action between the substances present in the hemolymph, which may contribute to a greater or lesser effect. This study describes too, for the first time, the evaluation of L. obliqua hemolymph and fraction on cell viability of neuronal cells primary culture. The results show that treatment of primary embryonic cultures of Wistar rats hipocampal neurons with CF for 24 h and subsequently exposure to H 2 O 2 for 30 min, showed increased cell viability compared to the positive control group, treated only with the inducing agent H 2 O 2 but they have significantly reduced mitochondrial metabolism when compared to the control without oxidative induction. The other conditions and treatment tested concentrations did not produce evidence that the hemolymph and fractions have produced a neuroprotective effect. Declarations Conflict of interest statement The authors have declared that there is no conflict of interest. Author Contribution Authors ContributionsSilviane Maggi(SM), Antônio Frederico Michel Pinto(AFMP), Mariana Sayuri Berto Udo(MSBU), Mariana Aguilera Alencar da Silva (MAS), Raphael Caio Tamborelli Garcia(RG), Luciane Minetto(LM), Leandro Tasso (LT), Pablo Machado(PM), Diógenes Santiago Santos(DSS), Paula Eichler(PE), Thiago Barcellos(TB), Pedro Ismael da Silva Junior(PISJR), Ronaldo Zucatelli Mendonça(RZM), Tania Marcourakis(TM) and Sidnei Moura(SiM)Names/Contributions: - Conceptualization and methodology: (SM, TM, SiM); - validation, formal analysis, investigation, resources, and data curation: (SM, AFMP, MSBU, MAS, RG, LM, LT, PM, DSS, PE, TB, PISJR, RZM, TM, SiM); - writing original draft: (SM); - review and editing, and visualization: (SM, AFMP, MSBU, MAS, RG, LM, LT, PM, DSS, PE, TB, PISJR, RZM, TM, SiM); - supervision: (RZM, TM, SiM); - project administration and funding acquisition: (RZM, TM, SiM). Acknowledgement The autors aknowledge the financial support by Brazilian agency CAPES. The autors gratefully aknowlege the support of Dr. Lisete M. Lorini, from University of Passo Fundo, Passo Fundo, Brazil, the Rio Grande do Sul Toxicology information Centre, Porto Alegre, Brazil, Dr. Mariana R. Ely and Dr. Edegar Fronza, from University of Caxias do Sul, Caxias do Sul, Brazil. References Alvarez-Flores MP, Remuzgo CM, Cury Y, Bosch RV, Vaz-de-Lima BB, Maria DA, Chudzinski-Tavassi AM (2012). Mechanisms implicated in cell proliferation and cell survival induced by recombinant Losac, a cell adhesion molecule from Lonomia oblique . Abstracts Toxins. Toxicon. 60:142-143. Basualdo A, Oliveira KC, Trevisan T, Barletta FB (2008). Tratamento clínico e bucomaxilofacial de quadro hemorrágico-alérgico por Lonomia sp: Relato de caso. Stomatos. 24(14):46-54. Bradford MM (1976). 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Hyg. 74(5):807-809. Maranga L, Mendonça RZ, Bengala A, Peixoto CC, Moraes RHP, Pereira CA, Carrondo MJT (2003). Enhancement of Sf-9 Cell Growht and Longevity through Supplementation of Culture Medium with Hemolymph. Biotechnol. Prog. 19:58-63. Mendonça RZ, Greco KN, Vieira H, Pereira ACP, Peixoto CC, Souza APB, Moraes RHP, Pereira CA, Carrondo MJT, Marques P. (2007). Participación de las mitocondrias em la acción anti-apoptótica de una proteína aislada de la hemolinfa de Lonomia obliqua . XII Congreso Nacional de Biotecnología y Bioingeniería, 2007, Morelia-Michoachacan-México. Annais del XII Congreso Nacional de Biotecnología y Bioingeniería. Morelia: Sociedad Mexicana de Biotecnología y Bioingeniería. 1:74-74. Mendonça RZ, Greco KN, Sousa APB, Moraes RHP, Astray RM, Pereira CA (2008). Enhancing effect of a protein from Lonomia obliqua hemolymph on ecombinant protein production. Cytotechnology. 57:83-91. Ortel J (1995). Changes in protein content and free amino acid composition in metal-contaminated gypsy moth larvae ( Lymantria dispar L., Lymantriidae, Lepidoptera). Comp. Biochem. Physiol. 112C(3): 291-298. Ratcliffe NA, Mello CB, Garcia ES, Butt TM, Azambuja P (2011). Insect natural products and processes: New treatments for human disease. Insect Biochem. Mol. Biol. 41:747-769. Rhee WJ, Park JH, Hahn J, Park TH (2013). Anti-apoptotic mechanism of silkworm hemolymph in HeLa cell apoptosis. Process Biochem. 48:1375-1380. Rhee WJ, Park TH (2000). Silkworm hemolymph inhibits baculovirus-induced insect cell apoptosis. Biochem. Biophys. Res. Commun. 271:186-190. Riella MC, Chula D, Freitas S, Mazza M, Pachaly MA (2008). Acute renal failure nd haemorrhagic syndrome secondaryto toxin of caterpillars ( Lonomia obliqua ). NDT Plus. 6:445-446. Roman H, Jegorov A (1991). Changes in free amino acid composition inhaemolymph of larvae of the wax moth, Galleria mellonella L., during cold acclimation. Comp. Biochem. Physiol. 100A(4):957-962. Silva RFM, Falcão AS, Fernandes A, Gordo AC, Brito MA, Brites D (2006). Dissociated primary nerve cell cultures as models for assessment of neurotoxicity. Toxicol Lett. 163:1-9. Souza APB, Peixoto CC, Maranga L, Carvalhal AV, Moraes RHP, Mendonça RMZ, Pereira CA, Carrondo MJT, Mendonça RZ (2005). Purification and characterization of an anti-apoptotic protein isolated from Lonomia obliqua hemolymph. Biotechnol Progr. 21:99-105. Veiga ABG, Ribeiro JMC, Guimarães JA, Francischetti IMB (2005). A catalog for the transcripts from the venomous structures of the caterpillar Lonomia obliqua : Identification of the proteins potentially involved in the coagulation disorder and hemorrhagic syndrome. Gene. 355:11–27. Vieira HLA, Pereira ACP, Peixoto CC, Moraes RHP, Alves PM, Mendonça RZ (2010). Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of Lonomia obliqua . Cytotechnology. 62:547-555. Wessel D, Flügge UI (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 138(1):141-143. Whitmore E, Gilbert LI (1974). Haemolymph proteins and lipoproteins in Lepidoptera – A comparative electrophoretic study. Comp. Biochem. Physiol. 47:63-78. Wilkins MR, Williams KL, Appel RD, Hochstrasser DF (Eds.) (1997). Proteome research : New frontiers in functional genomics. New York: Springer. 243p. Zannin M, Lourenço DM, Motta G, Dalla Costa LR, Grando M, Gamborgi GP, Noguti MA, Chudzinski-Tavassi AM (2003). Blood coagulation and fibrinolytic factors in 105 patients with hemorraghyc syndrome caused by accident contact with Lonomia obliqua caterpillar in Santa Catarina, Southern Brazil. Thromb. Haemost. 9(2):355-364. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx 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-5291061\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":369096994,\"identity\":\"6eeaed25-a1e4-41ba-80af-3993eeb41cc7\",\"order_by\":0,\"name\":\"Silviane Maggi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Caxias Do Sul\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Silviane\",\"middleName\":\"\",\"lastName\":\"Maggi\",\"suffix\":\"\"},{\"id\":369096996,\"identity\":\"6a4e1378-07f8-4538-8d87-4a4a16972bde\",\"order_by\":1,\"name\":\"Antonio Frederico Michel 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17:38:10\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-5291061/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-5291061/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":67375147,\"identity\":\"153bb5f4-d1fa-4be5-997b-18ae8fe2b32d\",\"added_by\":\"auto\",\"created_at\":\"2024-10-24 08:33:35\",\"extension\":\"jpeg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":182756,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eVenn diagram depicting the total number of proteins identified in 3 lots of hemolymph (H) and chromatographic fractions (CF) as well as the number of\\u003c/p\\u003e\\n\\u003cp\\u003eoverlapped proteins.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5291061/v1/cd222b8696d46fe2f3f6d43c.jpeg\"},{\"id\":67375150,\"identity\":\"8e20b29a-45cf-4380-8676-36cda208ff79\",\"added_by\":\"auto\",\"created_at\":\"2024-10-24 08:33:35\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":110355,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEvaluation of cell viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the three independents assays); H (Hemolymph; concentrations at 0.05% or 0.1% v/v); H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Positive control; concentrations at 10 or 30 µM). *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 and ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5291061/v1/eefa334e91961b6559227288.png\"},{\"id\":67376113,\"identity\":\"37117456-4400-45fd-93da-430cfed4cb88\",\"added_by\":\"auto\",\"created_at\":\"2024-10-24 08:41:35\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":130927,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEvaluation of celL viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the five independents assays); CF (Chromatographic fraction; concentrations at 0.05% or 0.1% v/v); H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Positive control; concentrations at 10 or 30 µM). *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 and ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 \\u0026nbsp;compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Onlinefloatimage7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5291061/v1/fee3f2051ed9b53577fcd04d.png\"},{\"id\":67970305,\"identity\":\"ce85f715-e22e-4e56-8cc7-44e40c3b89f4\",\"added_by\":\"auto\",\"created_at\":\"2024-10-31 21:16:28\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1071029,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5291061/v1/42a3ad61-05c2-4936-b43f-45dda8295a99.pdf\"},{\"id\":67375148,\"identity\":\"a50c962b-97b5-4513-8ba3-9bd3c605bac0\",\"added_by\":\"auto\",\"created_at\":\"2024-10-24 08:33:35\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":189482,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Table1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5291061/v1/21cec15c3673cbaafca61767.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Shotgun proteomic analysis of the caterpillar Lonomia obliqua (Lepitoptera, Saturniidae) hemolymph and effects in rat hippocampal neurons culture\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eInsects represent about 75% of all invertebrate animals, are among the more adapted to life on earth. These organisms are capable of producing a number of chemicals that help them survive the environmental attacks during their evolution (Ratcliffe \\u003cem\\u003eet al\\u003c/em\\u003e., 2011). Thus, evolutionarily, poisons and venoms animals were developed for the purpose of defense against predators or prey capture, causing physiological changes in natural enemies. However, they have potential application as new therapeutic drugs and have been the subject of research (Calvete, 2009). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eSeveral accidents involving the larval form of the \\u003cem\\u003eLonomia obliqua\\u003c/em\\u003e Walker, 1855 (Lepidoptera, Saturniidae) have been reported in southern Brazil, since the 80s (Duarte \\u003cem\\u003eet al.\\u003c/em\\u003e, 1990; Duarte \\u003cem\\u003eet al.\\u003c/em\\u003e, 1994; Kelen \\u003cem\\u003eet al.\\u003c/em\\u003e, 1995; Duarte \\u003cem\\u003eet al.\\u003c/em\\u003e, 1996; Zannin \\u0026nbsp;\\u003cem\\u003eet \\u0026nbsp; al.\\u003c/em\\u003e, \\u0026nbsp;2003). \\u0026nbsp;Individuals \\u0026nbsp; who \\u0026nbsp;had \\u0026nbsp;contact \\u0026nbsp; with \\u0026nbsp;bristles \\u0026nbsp;of \\u0026nbsp;\\u003cem\\u003eL. \\u0026nbsp; obliqua\\u003c/em\\u003e caterpillars can manifest a hemorrhagic syndrome associated with consumption coagulopathy which may include intravascular hemolysis and acute renal failure. Individual characteristics of the victims, intensity of exposure to poison and the number of animals involved, may determine slight, serious or even fatal accidents (Duarte \\u003cem\\u003eet al\\u003c/em\\u003e., 1996; Fan \\u003cem\\u003eet al\\u003c/em\\u003e., 1998; Gamborgi \\u003cem\\u003eet al\\u003c/em\\u003e., 2006; Malaque \\u003cem\\u003eet al\\u003c/em\\u003e., 2006; Riella \\u003cem\\u003eet al\\u003c/em\\u003e., 2008; Basulado \\u003cem\\u003eet al\\u003c/em\\u003e., 2008). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePhysiological activity presented by the venom in their victims initiated several studies on its composition, in order to develop therapy for poisoning and identify potential resources for the treatment of various conditions. Among the compounds identified a protein has been well characterized, called Lopap (\\u003cem\\u003eLonomia\\u003c/em\\u003e Prothrombin Activator Protease), but the literature still lacks studies to elucidate the overall protein content of venom composition and biological fluids caterpillar, which yet remains incomplete. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eProteomes are highly dynamic and may change with development and insect environment \\u0026nbsp; (Wilkins \\u0026nbsp;\\u003cem\\u003eet \\u0026nbsp;al\\u003c/em\\u003e., \\u0026nbsp;1997). \\u0026nbsp; Among \\u0026nbsp;the \\u0026nbsp;wide \\u0026nbsp; possibilities \\u0026nbsp;of \\u0026nbsp;bioactive molecules that insects can provide protein compounds with anti-apoptotic activity have been described (Rhee \\u003cem\\u003eet al.\\u003c/em\\u003e, 2013; Choi \\u003cem\\u003eet al.\\u003c/em\\u003e, 2002; Kim \\u003cem\\u003eet al\\u003c/em\\u003e., 2001; Rhee and Park, 2000). This activity may be interesting in pathologies that develop with cell loss by apoptosis, such as neurodegenerative. \\u003cem\\u003eL. obliqua\\u003c/em\\u003e hemolymph anti-apoptotic activity has been described for insect cells and mammals forward physical inducers, biological and chemical (Vieira \\u003cem\\u003eet al\\u003c/em\\u003e., 2010; Mendon\\u0026ccedil;a \\u003cem\\u003eet al\\u003c/em\\u003e., 2008; Souza \\u003cem\\u003eet al\\u003c/em\\u003e., 2005; Maranga \\u003cem\\u003eet al\\u003c/em\\u003e., 2003), but have not yet been described studies conducted with nerve cells. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThis study aimed identify hemolymph and fractions protein composition of three caterpillar colonies from different cities, collected at different times and feeding on various plants, and evaluate if there were significant changes between profiles of the same. Still, the effect of \\u003cem\\u003eL. obliqua\\u003c/em\\u003e caterpillars crude hemolymph and their chromatographic fractions was evaluated on cell viability of rat primary hippocampal cell cultures.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cem\\u003eHemolymph collection. Lonomia obliqua\\u003c/em\\u003e caterpillars was collected in state of Rio Grande do Sul (South of Brazil) and frozen at -20 \\u0026deg;C by 4 h. Hemolymph \\u0026nbsp;was harvested from sixty instar larvae after cutting bristles. The collected hemolymph was centrifuged by 14,000 rpm for 20 min, the supernatant was filtered through a 0.22 \\u0026micro;m membrane filter and stored at -20 \\u0026deg;C. \\u0026nbsp; \\u003cem\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eHemolymph Semi-Purified Fraction.\\u003c/em\\u003e Semi-purified fraction with anti-apoptotic activity was obtained from Butantan Institute (S\\u0026atilde;o Paulo, Brazil) (Souza \\u003cem\\u003eet al.\\u003c/em\\u003e, 2005). Briefly 0.5 mL of total hemolymph was fractionated on an AKTA Purifier chromatography system equipped with a Resource Q ion exchange column (Amersham Pharmacia Biotech, USA) at a rate of 0.5 mL min\\u003csup\\u003e-1\\u003c/sup\\u003e and eluted at a linear gradient (0-100%), TrisHCl 20 mmol L\\u003csup\\u003e-1\\u003c/sup\\u003e/Tris HCl \\u0026ndash; NaCl 1 mol L\\u003csup\\u003e-1\\u003c/sup\\u003e, pH 8.0. The eluate was monitored at 280, 254 and 214 nm and harvested in fractions of 1 mL. \\u0026nbsp; \\u003cem\\u003eAnimals.\\u003c/em\\u003e Wistar rats male (weighing 200 g, 100 days old) and virgin female (weighing 190 \\u0026nbsp; g, \\u0026nbsp;90 \\u0026nbsp;days old) \\u0026nbsp;were \\u0026nbsp; obtained \\u0026nbsp;from School of \\u0026nbsp;Pharmaceutical Sciences/Chemistry Institute, University of S\\u0026atilde;o Paulo, S\\u0026atilde;o Paulo, Brazil. Females were caged overnight with males and copulation was verified in the morning by detection \\u0026nbsp;of \\u0026nbsp;a \\u0026nbsp; vaginal \\u0026nbsp;plug. \\u0026nbsp;Pregnant \\u0026nbsp; females \\u0026nbsp;were \\u0026nbsp;housed \\u0026nbsp; under \\u0026nbsp;controlled temperature (22\\u0026plusmn;1 \\u0026deg;C) on a 12:12 hours light/dark cycle. Food and water were provided \\u003cem\\u003ead libitum\\u003c/em\\u003e. Fetuses were obtained at 18-19 days of pregnancy (E18-E-19). Pregnant rats were anesthetized with pentobarbital (45 mg Kg\\u003csup\\u003e-1\\u003c/sup\\u003e) and the fetuses were rapidly euthanized by decapitation to remove their hippocampi. All experiments were conducted according to the guidelines issued by the National Council for Animal Experimentation and this study was approved by the Animal Uses Ethic Committee of the Pharmaceutical Sciences School of University of S\\u0026atilde;o Paulo. \\u0026nbsp; \\u003cem\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eCell culture.\\u003c/em\\u003e Rat embryonic hippocampal primary cultures were obtained according Garcia \\u003cem\\u003eet al\\u003c/em\\u003e. (2012) with slightly modifications. Primary cultures were obtained by hippocampal neurons dissociation from hippocampi of E18-E19 Wistar rat embryos. Hippocampi were maintained in solution containing cooled neurobasal medium (Gibco, USA) with 100 U mL\\u003csup\\u003e-1\\u003c/sup\\u003e penicillin and 100 \\u0026micro;g mL\\u003csup\\u003e-1\\u003c/sup\\u003e streptomycin. Tissues were washed with \\u003cem\\u003eHank\\u0026rsquo;s Balanced Salt Solution\\u003c/em\\u003e (HBSS; Gibco, USA) and fragmented mechanically. \\u0026nbsp; Cell \\u0026nbsp;isolation \\u0026nbsp;was \\u0026nbsp; obtained \\u0026nbsp;by \\u0026nbsp;proteolytic \\u0026nbsp; digestion \\u0026nbsp;with \\u0026nbsp;trypsin according Jahr and Stevens (1987) and Silva \\u003cem\\u003eet al.\\u003c/em\\u003e (2006). Hippocampi fragments were incubated with trypsin 0,25% (Gibco, USA), pH 7.2-7.4, at 37 \\u0026deg;C for 10 min. Reaction was stopped with HBSS containing 277.5 U mL\\u003csup\\u003e-1\\u003c/sup\\u003e DNAse (Sigma, USA) and 10% fetal bovine serum (Gibco, USA), pH 7.2-7.4. Cells were then dispersed mechanically with Pasteur pipettes of different diameters. After, hippocampal cells were resuspended in neurobasal medium (Gibco, USA) supplemented with 0.5 mmol L\\u003csup\\u003e-1\\u003c/sup\\u003e L-glutamine (Gibco, USA), 25 \\u0026micro;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e L-glutamic acid (Sigma, USA), 100 U mL\\u003csup\\u003e-1\\u003c/sup\\u003e penicillin: 100 \\u0026micro;g mL\\u003csup\\u003e-1\\u003c/sup\\u003e streptomycin (Sigma, USA) and 2% B27 supplement (Gibco, USA), to reduce glial cell proliferation (Brewer \\u003cem\\u003eet al\\u003c/em\\u003e., 1993; Silva \\u003cem\\u003eet al\\u003c/em\\u003e., 2006). Cells suspension was then plated onto 0.01% poly-L-lysine-coated (Sigma, USA) 96-well culture plates at a density of 5.10\\u003csup\\u003e4\\u003c/sup\\u003e cells per well and incubated for 7-8 days at 37\\u0026deg;C, in humidified atmosphere of 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e for hippocampal neurons maturation. Half of the medium culture was replaced for the same volume of fresh medium with the same composition each 48 h. On 6-8 day, cultured cells were incubated with the treatments according to the experiment. Previous study by immunohistochemistry (Garcia \\u003cem\\u003eet al.\\u003c/em\\u003e, 2012) showed a predominance of neurons in this culture, with 92% of neurons and 8% of astrocytes. \\u0026nbsp; \\u003cem\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eApoptosis induction.\\u003c/em\\u003e Hippocampal cells (5.10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well) were treated with 10 and 30 \\u0026micro;mol \\u0026nbsp;L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026nbsp; solutions [from H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026nbsp; stock solution 30% (Merck, Germany), into supplemented neurobasal medium] fresh made, for 30 min (37\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e), after treatment with hemolymph and fractions in different concentrations. Exposure to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e solutions was performed within 5 minutes of dilution, in the dark. \\u003cem\\u003eCell viability.\\u003c/em\\u003e Cells viability was evaluated by MTT-reduction assay (Mosman, 1983; Liu \\u0026nbsp; et \\u0026nbsp; \\u0026nbsp; al., \\u0026nbsp;1997). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eActive \\u0026nbsp; metabolically \\u0026nbsp; \\u0026nbsp;cells \\u0026nbsp; \\u0026nbsp;are \\u0026nbsp; capable \\u0026nbsp; \\u0026nbsp;to \\u0026nbsp; cleave \\u0026nbsp; \\u0026nbsp; 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium \\u0026nbsp;bromide \\u0026nbsp; (MTT) \\u0026nbsp;by \\u0026nbsp;reduction \\u0026nbsp; of \\u0026nbsp;this yellow salt to formazan crystals, a purple compound, by mitochondrial reductases enzymes of viable cells. Formazan crystals can be measured by absorbance at 570 nm and directly correlated with viable cells number. For the assessment of cell viability, primary hipocampal cells in culture (5.10\\u003csup\\u003e4\\u003c/sup\\u003e cells/well; 7-8\\u0026deg; culture day) were incubated \\u0026nbsp;with \\u0026nbsp;hemolymph \\u0026nbsp; or \\u0026nbsp;fraction \\u0026nbsp;at \\u0026nbsp; 0.05 \\u0026nbsp;and \\u0026nbsp;0.1% \\u0026nbsp; (into \\u0026nbsp;supplemented neurobasal medium) for 1 and 24 h (37\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). After the period of incubation, cells were treated with 10 and 30 \\u0026micro;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026nbsp; solutions (into supplemented neurobasal medium) and incubated for 30 min (37\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). Treatments were then replaced by 100 \\u0026micro;L 0.5 mg mL\\u003csup\\u003e-1\\u003c/sup\\u003e MTT (Sigma-Aldrich Co., USA) solution and plates were incubated for 3 h (37\\u0026deg;C, 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e). Then, MTT solution was removed and 200 \\u0026micro;L of dimethyl sulfoxide (DMSO, Synth, Brazil) was added to each well. After, plates were shaking for 30 min and the absorbance was measured at 570 nm in a multiwell plate reader Synergy H1 Hybrid Reader (Biotek Instruments Inc., USA). \\u003cem\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eStatistical analysis.\\u003c/em\\u003e Data were reported as means \\u0026plusmn; SD (standard deviation) and statistical significance was evaluated by one-way analysis of variance (ANOVA) followed \\u0026nbsp;by \\u0026nbsp;the \\u0026nbsp; Newman-Keuls \\u0026nbsp;test \\u0026nbsp;(\\u003cem\\u003ep\\u003c/em\\u003e\\u0026nbsp; \\u0026lt; \\u0026nbsp; 0.05 \\u0026nbsp;was \\u0026nbsp;accepted \\u0026nbsp; as \\u0026nbsp;statistically significant). \\u0026nbsp; Experiments \\u0026nbsp;were \\u0026nbsp;conducted \\u0026nbsp; in \\u0026nbsp;quadruplicate \\u0026nbsp;from \\u0026nbsp; at \\u0026nbsp;least \\u0026nbsp;three independent experiments. Assays were analyzed by Prism 5 software (Graph Pad Software, USA). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.1 Shotgun analysis\\u003c/strong\\u003e \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eSample preparation.\\u003c/em\\u003e Protein concentration of the samples was determined using a Bradford assay (Bradford, 1976) with bovine serum albumin (BSA) as a standard. Proteins were concentrated according Wessel and Fl\\u0026uuml;gge (1984). Briefly, 20 \\u0026micro;g (total proteins) of each sample were submitted to protein precipitation with chloroform and methanol \\u0026nbsp; (1:3:1:3, \\u0026nbsp; sample:methanol:chloroform:destilated \\u0026nbsp; water) \\u0026nbsp;followed \\u0026nbsp;by centrifugation at 17,000 rpm, 10 min. Supernatant was removed and were added three volumes of methanol (centrifugation at 17,000 rpm, 10 min). After, liquid phase was \\u0026nbsp;discarded \\u0026nbsp;and \\u0026nbsp; precipitated \\u0026nbsp;was \\u0026nbsp;maintained \\u0026nbsp; at \\u0026nbsp;room \\u0026nbsp;temperature \\u0026nbsp; to \\u0026nbsp;dry. Precipitates of concentrated proteins were resuspended in buffer urea (8 mol L\\u003csup\\u003e-1\\u003c/sup\\u003e), tris (0.1 mol L\\u003csup\\u003e-1\\u003c/sup\\u003e) and was followed the digestion protocol adapted of Klammer and MacCoss (2006). Briefly, disulfide bonds were reduced by DTT 10 mmol L\\u003csup\\u003e-1\\u003c/sup\\u003e addition (37\\u0026ordm;C, 20 min) followed alkylation by IAA 50 mmol L\\u003csup\\u003e-1\\u003c/sup\\u003e addition (room temperature, in the dark, 20 min). After, urea was diluted to 2 mol L\\u003csup\\u003e-1\\u003c/sup\\u003e by 60 \\u0026micro;L of tris 0.1 mol L\\u003csup\\u003e-1\\u003c/sup\\u003e addition. Then, proteins were digested with trypsin (1:50, enzyme:substrate) in presence of CaCl\\u003csub\\u003e2\\u003c/sub\\u003e 1 mmol L\\u003csup\\u003e-1\\u003c/sup\\u003e (37\\u0026deg;C, 18 h). Reaction was stopped by formic acid addition (5% v/v, final concentration). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eNanoLC LTQ-XL Orbitrap MS/MS.\\u003c/em\\u003e Chromatography separations of tryptic peptide mixture was obtained by nanoLC Ultra (nanoLC Ultra 1D plus, Eksigent, USA) equipped with autosampler nanoLC AS-2 (Eksigent, USA) and connected to LTQ-XL Orbitrap Discovery MS/MS (Thermo Fischer Scientific, USA), containing a nano- electrospray ionization source (Thermo Fischer Scientific, USA). Analytical capillary columns (100 \\u0026micro;m \\u0026times; 20 cm) and pre-columns (150 \\u0026micro;m \\u0026times; 2 cm) was packaged \\u003cem\\u003ein\\u003c/em\\u003e \\u003cem\\u003ehouse\\u003c/em\\u003e with phase-reversed C18 (5 \\u0026micro;m ODS-AQ C18, Yamamura Chemical Lab). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHemolymph and chromatography fractions was loaded in autosampler with injection volume 10 \\u0026micro;L and at a flow rate of 1 \\u0026micro;L mL\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; for 15 min. Step gradient mobile \\u0026nbsp;phase A \\u0026nbsp;(5% \\u0026nbsp; acetonitrile, \\u0026nbsp;0.1% \\u0026nbsp;formic \\u0026nbsp; acid \\u0026nbsp;in \\u0026nbsp;water) \\u0026nbsp; was \\u0026nbsp;used \\u0026nbsp;to chromatography separations of 120 min (fractions) or 360 min (hemolymph) to mobile phase B (90% acetonitrile, 0.1% formic acid): 0-5% B in 5 min; 5-25% B in 60 min; 25-50% B in 20 min; 50-80% B in 15 min; 80% isocratic B for 5 min; 80-5% B in 1 min; 5% isocratic B for 14 min at a flow rate of 400 nL min\\u003csup\\u003e-1\\u003c/sup\\u003e). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePositive ion mode was employed and normalized collision energy was set to 35%. Full scan MS spectra were acquired from \\u003cem\\u003em/z\\u003c/em\\u003e 400-1,600 at a resolving power of 30,000, followed by acquisition of 8 MS2 mode spectra of the most abundant ions. Fragmentation was obtained by dissociated induced collision (CID), with \\u003cem\\u003eQ\\u003c/em\\u003e activation = 0.250, time of activation = 30 ms and isolation amplitude = 1 Da. Acquired peptide masses were added to dynamic exclusion list with size of 100 ions and time of permanence of 30 s during acquisition of MS2 spectra. Spray voltage used were 2.2 kV, capillary temperature of 275\\u0026deg;C, capillary voltage of 34 V, and the collision gas used was helium. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003e\\u0026nbsp;Data \\u0026nbsp; analysis.\\u003c/em\\u003e\\u0026nbsp; For protein identification, a no-redundant sequences data bank referred to entries of individuals proteins for \\u0026ldquo;\\u003cem\\u003eLonomia\\u003c/em\\u003e\\u0026rdquo; presents in NCBI data bank (http://www.ncbi.nlm.nih.gov) was constructed. From this bank, RAW files of MS-2 spectra was searched using \\u003cem\\u003eComet\\u003c/em\\u003e search software (Eng \\u003cem\\u003eet al.\\u003c/em\\u003e, 2013). Identification proteins and peptides was obtained using \\u003cem\\u003ePattern Lab for Proteomics\\u003c/em\\u003e software from homology with data bank sequences (Carvalho \\u003cem\\u003eet al\\u003c/em\\u003e., 2012). Candidates peptides were \\u0026nbsp; considered those containing one or \\u0026nbsp;two \\u0026nbsp;tryptic ends. Cysteine carbamidomethylation was adopted as fixed modification. Tolerance of 50 ppm to precursors ions and 1 Da to fragment ions were employed on the data search. \\u003cem\\u003e\\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eSearch Engine Processor\\u003c/em\\u003e software (Carvalho \\u003cem\\u003eet al.,\\u003c/em\\u003e 2012) was used to filter identified spectra. Parameters \\u003cem\\u003eXcorr\\u003c/em\\u003e, \\u003cem\\u003eDeltaCN\\u003c/em\\u003e, \\u003cem\\u003eDeltaMass\\u003c/em\\u003e, \\u003cem\\u003ePeaks Matched\\u003c/em\\u003e e \\u003cem\\u003eSpec\\u003c/em\\u003e \\u003cem\\u003eCount Score\\u003c/em\\u003e were used to generate a Bayesian score. Cutoff was established to accept 1% false-positive, based on reversed data base identifications. Furthermore, 6 residues were adopted as minimum sequence length. Results were post processed to set identifications less than 10 ppm of mass variation.\\u003c/p\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003e3.1 Hemolymph and chromatographic fraction protein identification by NanoLC\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMS/MS\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eStudies were conducted using a gel-free proteomic strategic in a nanoLC/LTQ- Orbitrap system. A total of 71 distinct hemolymph proteins were identified from 3 lots of caterpillars, with 34 common proteins between the same. In fractions was found 40 total distinct proteins, with 9 shared between the 3 lots. A Venn diagram displays the results in \\u003cstrong\\u003eFig. 1\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFig. 1 -\\u003c/strong\\u003e Venn diagram depicting the total number of proteins identified in 3 lots of hemolymph (H) and chromatographic fractions (CF) as well as the number of overlapped proteins. \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Considering hemolymph and CF samples, 76 different proteins were identified, with molecular weights ranging from 2,107.1 to 71,540.4 Da: 71 in hemolymph and 40 in CF, with 34 common proteins between then. Five proteins were found only in CF and 36 just in hemolymph.\\u003c/p\\u003e\\n\\u003cp\\u003eRelated to CF, a proportionately larger number of peptides was generated by hemolymph, due to the larger number of proteins present in this. The number of peptides produced by proteolytic digestion is proportional to abundance of its protein in a sample (Liu \\u003cem\\u003eet al.,\\u003c/em\\u003e 2004). Thus, peptides in larger quantity were selected to MS2 more frequently, generating a larger number of spectral counts which is proportional to the abundance of each protein in the sample. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFive proteins were identified only in CF, since in the crude sample, more peptides can compete to the selection of fragmentation within the same cycle of acquisition, and thus, peptides signal with less intensity can be suppressed at the expense of others compromising their identification. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eProtein identification in \\u003cem\\u003eL. obliqua\\u003c/em\\u003e samples by shotgun analysis is impaired by the limited range of data contained in the database for this specie. Proteins amino acids sequences data found in databases are mainly from transcriptomic studies (Veiga \\u003cem\\u003eet al.\\u003c/em\\u003e, 2005). \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eSerine proteases (45%), hemolins (38%), protease inhibitors (7%) and antiviral protein (5%) were found in CF, as well as sensory proteins, heat shock proteins, cysteine proteases and lectins less expressively. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn \\u0026nbsp;hemolymph \\u0026nbsp; were \\u0026nbsp;found \\u0026nbsp;predominantly \\u0026nbsp; antiviral \\u0026nbsp;protein \\u0026nbsp;(24%), \\u0026nbsp; serine proteases \\u0026nbsp;(23%), \\u0026nbsp;hemolins \\u0026nbsp; (16%) \\u0026nbsp;and \\u0026nbsp;protease \\u0026nbsp; inhibitors \\u0026nbsp;(10%). \\u0026nbsp;Heat \\u0026nbsp; shock proteins, serpins, \\u0026nbsp;sensory \\u0026nbsp; \\u0026nbsp; proteins, \\u0026nbsp; \\u0026nbsp;oxidoreductases, lectins, lyases and ribonucleases also were identified. Other found proteins together represent 20% of the total, and are structural proteins or proteins involved in metabolic functions. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eSignificant qualitative and quantitative variations in hemolymph amino acids concentration and protein composition were related to insect age and stage of development (Roman and Jegorov, 1991; Whitmore and Gilbert, 1974), diet (Ortel, 1995) and ambient temperature (Roman and Jegorov, 1991; Cui \\u003cem\\u003eet al.\\u003c/em\\u003e, 2011). Thus, this can have determined differences in protein composition observed in the three lots of hemolymph. \\u0026nbsp; \\u0026nbsp; \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1 -\\u003c/strong\\u003e Identified proteins on hemolymph and chromatographic fractions samples by \\u003cem\\u003eshotgun\\u003c/em\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;Insects present a metamorphosis process where a combination of grow\\u0026nbsp; sinaling, activation, differentiation and specific tissues physiological apoptosis is\\u0026nbsp; highly regulated. Probably substances responsible for this signaling circulate in\\u0026nbsp; hemolymph, which makes their interesting for study to identification of bioactive\\u0026nbsp; compounds with biotechnological potential (Maranga \\u003cem\\u003eet al.\\u003c/em\\u003e, 2003). \\u0026nbsp;\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003e3.2 Hemolymph and CF effects on H\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003eO\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e\\u003csub\\u003e2\\u003c/sub\\u003e\\u003c/em\\u003e\\u003c/strong\\u003e\\u003cstrong\\u003e\\u003cem\\u003e- treated cells\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2.1 Hemolymph\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e Mitochondrial metabolism of 0.05% and 0.1% hemolymph treated cells for 1 h and subsequently subjected to apoptosis induction with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026nbsp; 10 \\u0026nbsp; \\u0026mu;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; \\u0026nbsp;was significantly reduced (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01) compared to control without inducing apoptosis.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eHowever, \\u0026nbsp;when compared to apoptosis induction control with \\u0026nbsp;the same H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u0026nbsp;\\u003c/sub\\u003econcentration, there was no significant difference. Viability of 0.05% and 0.1% hemolymph cells treated for 1 h with apoptosis induced by H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30 \\u0026nbsp; \\u0026mu;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; also had a significant reduction (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001) relative to untreated control, but there was no significant difference when the comparison was made with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30 \\u0026nbsp;\\u0026mu;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; control. Treatment with 0.05% hemolymph, H2O2 10 \\u0026nbsp; mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; showed no statistical difference compared to control and H2O2 \\u0026nbsp;10 \\u0026mu;molL\\u003csup\\u003e-1\\u003c/sup\\u003e. Cells submitted to 0.1% hemolymph, H2O2 10\\u0026nbsp;\\u0026mu; mol\\u0026nbsp;L\\u003csup\\u003e-1\\u003c/sup\\u003e had significant reduction (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001) in cell viability compared to control and H2O2 10\\u0026mu;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05).\\u003c/p\\u003e\\n\\u003cp\\u003eCells with 0.05% and 0.1% hemolymph for 24 h, exposed to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30mol L\\u003csup\\u003e-1\\u0026nbsp;\\u003c/sup\\u003ehad significantly reduction viability (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001) relative to control and was not difference in comparison with H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30 \\u0026nbsp;\\u0026mu;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003ePresence of other active substances that may play an inhibitory effect on cell viability and proliferation, such as A2 phospholipase involved in hemolytic activity presented \\u0026nbsp;by \\u0026nbsp;the \\u0026nbsp; poison \\u0026nbsp;and \\u0026nbsp;hyaluronidase \\u0026nbsp; activity \\u0026nbsp;should \\u0026nbsp;be \\u0026nbsp; considered \\u0026nbsp;in evaluating the crude hemolymph effect (Heinen \\u003cem\\u003eet al\\u003c/em\\u003e., 2014). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFig. 2 -\\u003c/strong\\u003e Evaluation of cell viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the three independents assays); H (Hemolymph; concentrations at 0.05% or 0.1% v/v); H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Positive control; concentrations at 10 or 30 \\u0026micro;M). *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 and ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison). \\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2.2 \\u0026nbsp; Chromatographic Fraction (CF)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTreated cells with 0.05% and 0.1% CF for 1 h and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 10mol L\\u003csup\\u003e-1\\u0026nbsp;\\u003c/sup\\u003eproduced viable cells significant reduction (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 and \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 respectively) compared to untreated control and 0.05% and 0.1% CF for 1 h, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2 30 mol\\u0026nbsp;\\u003c/sub\\u003eL\\u003csup\\u003e-1\\u0026nbsp;\\u003c/sup\\u003e(\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001). There were no significant differences when comparing CF and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2 10\\u0026nbsp;\\u003c/sub\\u003emol\\u0026nbsp;L\\u003csup\\u003e-1\\u0026nbsp;\\u003c/sup\\u003eeither between CF and H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30mol L\\u003csup\\u003e-1\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eFor 0.05% and 0.1% CF (24 h),H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e\\u0026nbsp; 10 \\u0026nbsp;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e\\u0026nbsp; there is not statistical difference compared to control, however occurred cell viability increase related to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 10 \\u0026nbsp;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 and \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01, respectively). Cells with 0.05%and 0.1% for24 h, H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e 30 \\u0026nbsp;mol L\\u003csup\\u003e-1\\u003c/sup\\u003e had significant reduction viability (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01) regarding control.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIncrease in viable cells (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05) was produced by 0.05% CF, H2O2 30 \\u0026mu;mol L-1 related to H2O2 30 \\u0026mu;mol L-1 while 0.1% CF, H2O2 30 \\u0026mu;mol L-1 had no difference. \\u0026nbsp; \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;\\u003cstrong\\u003eFig. 3 -\\u003c/strong\\u003e Evaluation of celL viability by MTT assay. CTRL (Control; n = 5 wells per group in each of the five independents assays); CF (Chromatographic fraction; concentrations at 0.05% or 0.1% v/v); H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e (Positive control; concentrations at 10 or 30 \\u0026micro;M). *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01 and ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001 \\u0026nbsp;compared with CTRL and intergroup comparison (ANOVA and Newman-Keuls multiple comparison). \\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eAfter 24 h of exposure the rat hipocampal neurons culture to CF, and subsequently exposure to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e for 30 min, the cellular viability kept higher than positive control (only H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e), but a reduction in neuronal viability was observed related to control (without oxidative induction).\\u003c/p\\u003e\\n\\u003cp\\u003eSeveral \\u0026nbsp;studies \\u0026nbsp; have \\u0026nbsp;shown \\u0026nbsp;the \\u0026nbsp; property \\u0026nbsp;of \\u0026nbsp;substances \\u0026nbsp; from \\u0026nbsp;insects hemolymph in the inhibition of apoptosis promoted by different inducers (physical, chemical and biological), in insects and mammals cell cultures (Rhee \\u003cem\\u003eet al.\\u003c/em\\u003e, 2013; Vieira \\u003cem\\u003eet al.\\u003c/em\\u003e, 2010; Mendon\\u0026ccedil;a \\u003cem\\u003eet al.\\u003c/em\\u003e, 2008; Souza \\u003cem\\u003eet al.\\u003c/em\\u003e, 2005; Maranga \\u003cem\\u003eet al.\\u003c/em\\u003e, 2003; Choi \\u003cem\\u003eet al.\\u003c/em\\u003e, 2002; Kim \\u003cem\\u003eet al.\\u003c/em\\u003e, 2001; Rhee and Park, 2000). The effect in a variety of cells opposite the apoptosis induction by multiple mechanisms suggests that it may act in some apoptosis conserved way (Heinen \\u003cem\\u003eet al.\\u003c/em\\u003e, 2014).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eL. obliqua\\u003c/em\\u003e hemolymph anti-apoptotic activity was observed in Sf-9 insect cells (Souza \\u003cem\\u003eet al.\\u003c/em\\u003e, 2005; Maranga \\u003cem\\u003eet al\\u003c/em\\u003e., 2003; Vieira \\u003cem\\u003eet al.\\u003c/em\\u003e, 2010), in HEK-293 human cells (Mendon\\u0026ccedil;a \\u003cem\\u003eet al\\u003c/em\\u003e., 2007) and in mammalian cells V-79 (Heinen \\u003cem\\u003eet al.\\u003c/em\\u003e, 2014). Mendon\\u0026ccedil;a \\u003cem\\u003eet al\\u003c/em\\u003e. (2007) observed that exposure to hemolymph and chromatographic fractions produced high electrochemical potential in the mitochondrial membrane maintenance. Cytoprotective activity was described too in leucocytes and endothelial cells (Chudzinski-Tavassi \\u003cem\\u003eet al.,\\u003c/em\\u003e 2010), in HUVECs (Fritzen \\u003cem\\u003eet al.,\\u003c/em\\u003e 2005) and in fibroblasts FN-1 (\\u003cem\\u003erLosac \\u0026ndash;\\u003c/em\\u003e recombinant \\u003cem\\u003eLonomia obliqua\\u003c/em\\u003e Stuart-Factor Activator) (Alvarez-Flores \\u003cem\\u003eet al.\\u003c/em\\u003e, 2012), but associated to different proteins from \\u003cem\\u003eL. obliqua\\u003c/em\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFurther studies to verify expressed proteins from samples treated cultures may indicate if factors such as cells from animals tissues adaptation differences to \\u003cem\\u003ein vitro\\u0026nbsp;\\u003c/em\\u003econdition can be related to results obtained. Moreover, it is still unknown anti-apoptotic protein interaction with specific cellular receptors that may not be present or not be available in quantities sufficient for the manifestation of the effects in the studied cell. The structure of the anti-apoptotic effects responsible protein remains without being elucidated and thus the prediction of their susceptibility to degradation or inactivation are also compromised.\\u003c/p\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003eAs demonstrated in this study, animal venoms protein composition such as \\u003cem\\u003eL. obliqua\\u003c/em\\u003e can provide qualitative and quantitative variations intra species when Compared different origins animals and consequently exposed to various environmental factors. Concentration of free ions naturally present in the hemolymph can likewise differ between animals and produce shift in the elution of specific proteins during ion exchange chromatography making not reproducible elution pattern of the fractions. Thus, proteins present in fraction used in this study may differ over those described in the literature using the same fractionation strategy. Still, there may be synergistic action between the substances present in the hemolymph, which may contribute to a greater or lesser effect.\\u003c/p\\u003e\\u003cp\\u003eThis study describes too, for the first time, the evaluation of \\u003cem\\u003eL. obliqua\\u003c/em\\u003e hemolymph and fraction on cell viability of neuronal cells primary culture. The results show that treatment of primary embryonic cultures of Wistar rats hipocampal neurons with CF for 24 h and subsequently exposure to H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e for 30 min, showed increased cell viability compared to the positive control group, treated only with the inducing agent H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e but they have significantly reduced mitochondrial metabolism when compared to the control without oxidative induction. The other conditions and treatment tested concentrations did not produce evidence that the hemolymph and fractions have produced a neuroprotective effect.\\u003c/p\\u003e \"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eConflict of interest statement\\u003c/h2\\u003e \\u003cp\\u003eThe authors have declared that there is no conflict of interest.\\u003c/p\\u003e \\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eAuthors ContributionsSilviane Maggi(SM), Ant\\u0026ocirc;nio Frederico Michel Pinto(AFMP), Mariana Sayuri Berto Udo(MSBU), Mariana Aguilera Alencar da Silva (MAS), Raphael Caio Tamborelli Garcia(RG), Luciane Minetto(LM), Leandro Tasso (LT), Pablo Machado(PM), Di\\u0026oacute;genes Santiago Santos(DSS), Paula Eichler(PE), Thiago Barcellos(TB), Pedro Ismael da Silva Junior(PISJR), Ronaldo Zucatelli Mendon\\u0026ccedil;a(RZM), Tania Marcourakis(TM) and Sidnei Moura(SiM)Names/Contributions: - Conceptualization and methodology: (SM, TM, SiM); - validation, formal analysis, investigation, resources, and data curation: (SM, AFMP, MSBU, MAS, RG, LM, LT, PM, DSS, PE, TB, PISJR, RZM, TM, SiM); - writing original draft: (SM); - review and editing, and visualization: (SM, AFMP, MSBU, MAS, RG, LM, LT, PM, DSS, PE, TB, PISJR, RZM, TM, SiM); - supervision: (RZM, TM, SiM); - project administration and funding acquisition: (RZM, TM, SiM).\\u003c/p\\u003e\\u003ch2\\u003eAcknowledgement\\u003c/h2\\u003eThe autors aknowledge the financial support by Brazilian agency CAPES. The \\nautors gratefully aknowlege the support of Dr. Lisete M. Lorini, from University of \\nPasso Fundo, Passo Fundo, Brazil, the Rio Grande do Sul Toxicology information Centre, Porto Alegre, Brazil, Dr. Mariana R. Ely and Dr. Edegar Fronza, from University of Caxias do Sul, Caxias do Sul, Brazil.\\n\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eAlvarez-Flores MP, Remuzgo CM, Cury Y, Bosch RV, Vaz-de-Lima BB, Maria DA, Chudzinski-Tavassi AM (2012). Mechanisms implicated in cell proliferation and cell survival induced by recombinant Losac, a cell adhesion molecule from \\u003cem\\u003eLonomia oblique\\u003c/em\\u003e. Abstracts Toxins. \\u003cem\\u003eToxicon.\\u003c/em\\u003e 60:142-143.\\u003c/li\\u003e\\n \\u003cli\\u003eBasualdo A, Oliveira KC, Trevisan T, Barletta FB (2008). 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Improvement of recombinant protein production by an anti-apoptotic protein from hemolymph of \\u003cem\\u003eLonomia obliqua\\u003c/em\\u003e. \\u003cem\\u003eCytotechnology.\\u003c/em\\u003e 62:547-555.\\u003c/li\\u003e\\n \\u003cli\\u003eWessel D, Fl\\u0026uuml;gge UI (1984). A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. \\u003cem\\u003eAnal Biochem.\\u003c/em\\u003e 138(1):141-143.\\u003c/li\\u003e\\n \\u003cli\\u003eWhitmore E, Gilbert LI (1974). Haemolymph proteins and lipoproteins in Lepidoptera \\u0026ndash; A comparative electrophoretic study. \\u003cem\\u003eComp. Biochem. Physiol.\\u003c/em\\u003e 47:63-78.\\u003c/li\\u003e\\n \\u003cli\\u003eWilkins MR, Williams KL, Appel RD, Hochstrasser DF (Eds.) (1997). \\u003cem\\u003eProteome\\u0026nbsp;\\u003c/em\\u003e\\u003cem\\u003eresearch\\u003c/em\\u003e: New frontiers in functional genomics. New York: Springer. 243p.\\u0026nbsp;\\u003c/li\\u003e\\n \\u003cli\\u003eZannin M, Louren\\u0026ccedil;o DM, Motta G, Dalla Costa LR, Grando M, Gamborgi GP, Noguti MA, Chudzinski-Tavassi AM (2003). Blood coagulation and fibrinolytic factors in 105 patients with hemorraghyc syndrome caused by accident contact with \\u003cem\\u003eLonomia obliqua\\u003c/em\\u003e caterpillar in Santa Catarina, Southern Brazil. \\u003cem\\u003eThromb. Haemost.\\u0026nbsp;\\u003c/em\\u003e9(2):355-364.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"},{\"header\":\"Tables\",\"content\":\"\\u003cp\\u003eTable 1 is available in the Supplementary Files section.\\u003c/p\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"Lonomia, shotgun, proteomic, apoptosis, hippocampal culture\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5291061/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5291061/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eStudy of substances with potentially neuroprotective has been one of the research focus on drugs development. Toxic proteins of \\u003cem\\u003eLonomia obliqua\\u003c/em\\u003e caterpillars, which have caused several accidents in southern Brazil, were identified in the hemolymph with anti-apoptotic activity. This study aims the evaluation of the protein profile and the hemolymph effect on cell viability of rats’ primary cultured hippocampal neurons after apoptosis induction. Semi-quantitative shotgun proteomics approach was used to evaluate the protein profile of 3 caterpillars lots of different origin. Were identified a total \\u0026nbsp;of \\u0026nbsp; 76 \\u0026nbsp;proteins, \\u0026nbsp;71 \\u0026nbsp; in hemolymph and 40 in fractions. Antiviral protein predominated in crude hemolymph, following by serine proteases, hemolins and protease inhibitors. In fractions were identified hemolins, serine proteases and protease inhibitors. The treatment of rats’ primary cultured hippocampal neurons with the chromatographic fraction at concentration of 0.05 and 0.10% (v/v) for 24 hours, with subsequently apoptosis induction was able to maintain cell viability significantly higher than positive control. Hemolymph protein composition can show qualitative and quantitative variations intra species when compared different origins animals and consequently exposed to various environmental factors. The results shown on this study may contribute to the identification of proteins with potential use as neuroprotective in degenerative conditions.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Shotgun proteomic analysis of the caterpillar Lonomia obliqua (Lepitoptera, Saturniidae) hemolymph and effects in rat hippocampal neurons culture\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-10-24 08:33:30\",\"doi\":\"10.21203/rs.3.rs-5291061/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"d3c6b3ef-c569-4096-9fef-cb53be4bba2b\",\"owner\":[],\"postedDate\":\"October 24th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-11-02T12:53:07+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-10-24 08:33:30\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5291061\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5291061\",\"identity\":\"rs-5291061\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}