In-silico analysis of FASLG gene resistance in Cancer Immunotherapy

In-silico analysis of FASLG gene resistance in Cancer Immunotherapy | 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 In-silico analysis of FASLG gene resistance in Cancer Immunotherapy Shouhartha Choudhury This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6582476/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 Background The FASLG gene in the TNF superfamily binds with the receptor superfamily to induce apoptosis. FAS ligand and receptor interactions lead to a dominant nature in the immune process and control cellular death (apoptosis). The dynamism of the apoptotic-induced FASLG gene controls tumour-infiltrating lymphocytes and suppresses tumour responses called tumour counter-attacks. Intense evidence in the animal model illustrated that the FASLG (CD95L) gene functions govern T-cell revival and tumour exit. The justification of CD95L (CD178) controls tumours and induces inflammation. Those mechanisms forwarded the T-cell back into the tumour context by immune checkpoints restraining tumour growth. Also, the mechanisms suggested that CD178 in tumour cells contributes to immune escape. Objective So, the study proposed an in-silico analysis of the FASLG gene and its TNF family to pursue the molecular immunologic mechanisms linked with cancer and immunity in the model organisms. Hence, the study utilized bioinformatics and computational applications to learn about the TNF domain-mediated genes in two isolated organism’s genome. This application proposed a significant way to study particular genes in the genome. Results A genome-wide observation suggested multiple hits of the TNF domains in the fundamental TNF superfamily in Homo sapiens and Mus musculus . Further observation of the FASLG gene demonstrated the nucleotides, peptides, 3D structure, domain, motifs, phylogeny, gene expression, gene network, chromosome location, and signalling pathway in humans. The study remarked that the FASLG gene and its functional mechanisms are integrated with the immune process. Concluding remarks: The documented data forward the FALSG gene and its molecular mechanisms during T-cell initiation in the tumour microenvironment. Those molecular mechanisms proposed T-cell suppression by immune checkpoints and control of tumour progression via anti-tumour immune response. FASLG TNF Family Cancer Gene Therapy Immunotherapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction In the 19th century, William Coley proved that the tumours in some sarcoma patients contracted infections were triggered to necrotize. Further, it was discovered that lipopolysaccharides (LPS) are responsible for initiating the tumor-necrotizing activity. However, the biochemically and genetically described factor in serum provokes tumours to regulate molecules was proposed as a “tumour necrotizing factor” in 1975. This molecule, later called “tumour necrosis factor”, is a created component of a superfamily of closely related proteins [ 1 – 3 ]. Several reviews and data suggested that the classical TNF superfamily components are assumed through the plasma membrane via proteolytic cleavage (proteolysis) and represent a cytokine. Also, the fundamental nature of the superior TNF superfamily folds with the TNFR superfamily as a cytokine by the cysteine-rich residue. The TNFSF/TNFRSF ultimately includes 19 ligands & 29 receptors, revealing essential phenomena in the cellular process. So, the TNF superfamily is identified based on its gene sequence and functions [ 4 ]. TNF-alpha and TNF-beta are first observed at the peptide level before isolation of their cDNA. Also, other components in the TNF superfamily were identified by cDNA sequence. The fundamental TNF superfamily includes TNF-alpha, TNF-beta, FASL, LT-beta, CD40L, CD27L, CD30L, 4-1BBL, RANKL, TRAIL, LIGHT, TWEAK, OX40L, APRIL, BAFF, GITRL, EDA-A1, EDA-A2, and VEGI. These members are binding to the 29 multiple TNF receptors. However, the terminology of the major TNF superfamily has ligands and receptors that are generalized by the International Commission. The TNF ligands are observed by the antigen-presenting cells, like dendritic cells, B lymphocytes and macrophages in the immune process. Also, the TNF ligands are standardized by NK cells, T lymphocytes, eosinophils, basophils, mast cells, epithelial cells, endothelial cells, and muscle cells. The signals initiated by TNFSF/TNFRSF are variable for homeostasis and growth in organisms [ 5 ]. Also, TNFSF/TNFRSF components govern cellular adaptation, survival, and apoptosis, and their critical functions sustain immunity. The innate and adaptive immunity governed by TNFSF/TNFRSF components are valuable for the harmony of variant mechanisms leading to co-stimulation/co-inhibition. Malfunctions of the TNFSF/TNFRSF signalling are involved towards autoimmune and inflammatory disorders and underscore the magnitude of gene regulation. So, the concept of the TNFSF/TNFRSF activities led to decreased chronic inflammation and promoted anti-tumour immunity. Thus, the significant mechanisms underscore the molecular signalling that mediates the design and development of efficient anti-inflammatory and anti-cancer therapies [ 6 – 9 ]. Further concept of the TNF/NGF family reveals cognate receptors control immune functions, tissue homeostasis, and embryonic growth. So, the TNF/NGF family contributes to the decision of lymphocytes in the thymus. TNFR1, TNFR2, OX40, CD27, DR3, CD30, HVEM, 4-1BB, BAFF, TACI, and further transmembrane activators provide signals in antigen-stimulated T cells. Those receptors initiate stimulatory signals to enhance lymphocyte maturation and survival. These diverse natures are ordered by shared properties of family components by interactions of various factors with non-family ligands and co-receptors. The spectra are further prolonged by the phenomenon of ligands and receptors towards membrane-soluble by ensuring soluble formation to the extracellular matrix via signalling through intracellular domains [ 10 ]. So, the major TNF superfamily is shaped by a homology domain that forms trimers. The classical tumour necrosis factor superfamily is a peptide family separated into two membrane polypeptides in the mammalian genome. The peptides in the classical TNF superfamily are conserved and located in the beta-sheet. The homology residue of the supreme TNF superfamily appears through molecular checkpoints and controls cellular functions (i.e. differentiation, proliferation, invasion, angiogenesis, embryogenesis, immune response, inflammation, and cellular death) [ 9 ]. So, the defensive TNF superfamily appears in immunologic, neurologic, myologic, pulmonary, and metabolic diseases. Concerning cancer therapy, studies in the late 1980s explored how cancer cell mortality was affected when treated through TNF, and the TNF provision of cohorts led to an inflammatory shock syndrome [ 5 ]. TNF is a dynamic proinflammatory molecule, and its blocking activity has proved to be efficacious in decreasing the inflammation linked with autoimmunity. Immune-initiated mechanisms of tumours include antigen presentation and apoptosis resistance by active strategies and the production of immunosuppressive paracrine factors [ 8 ]. Thus, CD95-induced selection of B cells is required for the homeostasis of both T and B cells. Studies of DC-specific CD95-deficient showed that CD95 plays a key role in DC homeostasis by controlling the immunogenicity of self-antigen presentation. Furthermore, CD95-mediated elimination of autoreactive T cells prevents the autoimmunity. So, the phenotypes observed by the lacking CD95L in T or B cells in organism. However, loss of CD95L specifically in CD4 + and CD8 + T cells did not affect the concentration of the T-cell components during illness. These results led to CD95L in T cells and homeostasis in response to acute stimuli, but are required to prevent autoimmunity mediated by chronically activated T cells. So, the inactivation of CD95 in T cells was accompanied by lymphopenia, because CD95L levels on activated T cells led to the apoptosis of peripheral lymphocytes.Thus, many tumours proved to reveal the apoptosis-inducing cell surface molecule called CD178 (CD95L, FASL, and APO-1L). The CD95L response can govern the maturation of killer T cells in an antigen-specific fashion. Also, immune reactions are prevented by major histocompatibility complex (MHC) and secretion of immunosuppressive cytokines. These mechanisms facilitate cancer growth in the immune process and have proven to be an immunotherapy [ 11 – 15 ]. Immune therapy depends on several molecular functions and mechanisms that may promote tumour growth through the immune process. The immunologic theorem proposed that cancer immunotherapy builds on the molecular immune checkpoints that control T-cell activation. Those molecular mechanisms foster therapeutic applications in immuno-oncology [ 11 ]. Thus, the study suggested an in-silico analysis of the FASLG gene from the TNF superfamily in mammalian genomes. The observation of TNF domain-mediated genes in the TNF superfamily is mandatory to explore the molecular function and mechanisms associated with T-cell immunity. So, perform bioinformatics and computational techniques to examine the TNF domain-initiated genes in the powerful TNF superfamily in two mammalian genomes. Results Structural analysis The primary sequence determined the concentration of nucleotides and peptides in the FASLG gene in Homo sapiens . Also, the sequence arranged by 846 nucleotides and 281 peptides among 137 peptides binds to DNA ( Table 1: Primary sequence of FASLG gene ). The nature of the FASLG is stable and forms homotrimers. Also, each promoter contains two beta-sheets and a conserved proline-rich residue employed in major intracellular transport and signalling ( Fig. 1: 3D structure of FASLG ). Genome-wide analysis The genome assembly determined the two model organisms of Homo sapiens and Mus musculus . Further, the genome-wide examination of both organisms proposed the HMMER algorithm and obtained 47 and 15 TNF domains in Homo sapiens and Mus musculus , respectively ( Table 2: Summary of the (a) TNF domain (b) Homologs, and (c) FASLG ). Standalone BLAST2 outcome represents 43 and 14 homologs of FASLG genes in Homo sapiens and Mus musculus , respectively ( Table 2: Summary of the (a) TNF domain (b) Homologs, and (c) FASLG ). The gene ontology (GO) annotation confirmed the sequence accuracy of the TNF domain-mediated FASLG gene in Homo sapiens and Mus musculus ( Table 3: Summary of the genes in the TNF superfamily ). Domain, motifs, and phylogeny analysis The highest hits of the CD95L (FASLG) were listed from both organisms for sequence alignment. The MSA results demonstrated a conserved TNF domain in FASLG (CD178) genes in both organisms. The ratio of consensus (50%) confirmed the extended TNF domain ( Fig. 2: Conserved TNF domain in FASLG gene ) and its specific motifs ( Fig. 3: Sequence-specific motif in FASLG genes ) in the CD178 (CD95L). So, the observation concluded that the TNF domain is conserved in evolution. The phylogenetic tree suggested that the molecular evolutionary link of the TNF domain-mediated genes in-between Homo sapiens and Mus musculus ( Fig. 4: Molecular evolutionary link between FASLG and TNF-domain mediated genes in genomes ). Chromosome location, gene network, gene expression, and pathway analysis Further, five categories of human mRNA explore one measure of the FASLG expression medium (log2 scale: 0.1) in the neoplasms of the eye, brain, central nervous system, and glioblastoma ( Fig. 5: Expression analysis of FASLG gene in human ). A chromosome location study confirmed that the FASLG gene is located at band 1q24.3. Started at 172,659,103 bp and stopped at 172,666,876 bp in humans ( Fig. 6: FASLG gene located at Chromosome 1 in human ). The gene network study determined that the CD178 interacts with other molecules such as CASP10, CASP8, FOXO3, TNFRSF6B, TNFRSF1A, FAS, FADD, CFLAR, BID, and ENSG00000026036. Those molecular interactions govern the outcome of the CD95L in the cellular process ( Fig. 6: FASLG gene interacts with other TFs in cellular process ). Finally, the pathway study concluded the FASLG signalling in cellular death ( Fig. 8: FASLG signalling in cellular death ). Discussion The defensive immune system promises to be a key component of optimal cancer therapy. Effective strategies elicit immune replication and control tumours by enabling tumour-reactive T lymphocytes to infiltrate tumours. Cancer has dynamic levels of intra-tumoral T lymphocytes that significantly enhance survival across multiple tumours [ 8 , 12 , 13 ]. So, T-cell aggression is essential for anti-tumour immunity and tumour mortality. Tumours employed complex biological phenomena linked with immune evasion and angiogenesis. Also, tumour angiogenesis is coordinated with the depth of T cell-mediated tumour rejection. So, the angiogenesis is perturbed by the conflict of the tumour endothelium. The tumour endothelium imposes substantial limits on T lymphocyte penetration, leading to tumour endothelial blockage. Thus, successful immunotherapy is based on molecular strategies to control the tumour endothelium. Consequently, the tumour endothelial barrier is coordinated by endothelial-T cells and governs T lymphocyte trafficking [ 14 – 21 ]. However, a classical mechanism of dynamic pro-angiogenic growth factor of the VEGF-A governs endothelial-T cells by releasing VCAM-1 and intercellular resistance molecules (i.e. ICAM-1, ICAM-2, ICAM-3, ABCC1, and ABCB1) in endothelial cells. In contrast, the ABCDS (endothelin B receptor or ETBR) is linked with vascular concern and further controls T cell commitment to the endothelium. The response of VEGF-A/ETBR increases the rate of T-lymphocyte infiltration in tumours and promotes immune therapy [ 8 , 22 – 25 ]. Also, VEGF-A with antibody (G6-31) and cyclooxygenase enzymes with acetylsalicylic acid (ASA) were effective in inducing tumour-infiltrating CD8 + T cells. The frequency of CD8 + cells in tumours was coordinated with the ratio of FASLG-positive vessels. Immune-initiated mechanisms were induced by FASL through T cell receptors in tumours and treated by VEGF-A-specific antibody and acetylsalicylic acid. However, granzyme-B (GrB) and interferon-γ (IFN-γ) respond in tumours treated by VEGF-A-specific antibodies and acetylsalicylic acid. Thus, ASA and VEGF-A-specific antibodies are treated by endothelial FASL, enabling CD8 + T lymphocyte infiltration in tumours. So, the IFN-γ and granzyme-B were linked with a ratio across tumours and treated by VEGF-A specific antibody and acetylsalicylic acid, suggesting cytotoxic T lymphocyte-initiated tumour suppression [ 8 ]. The mobilization of anti-tumour immunity, and lack of CD8 + cells, but not CD4 + cells, governs the ability of the VEGF-A-specific antibody and acetylsalicylic acid combination to inhibit tumour growth. Interestingly, CD4 + cells eliminated tumours and enhanced anti-tumour immunity. Numerous mechanisms are forwarded by the endothelium, revealing a critical function through T-cell groups [ 22 , 26 , 27 ]. However, the FASLG is a conventional homeostatic mediator of T cell and apoptosis and manifests in a tumour endothelium. FASLG is observed by vasculature and the penetration of pro-angiogenic and immunosuppressive paracrine factors (i.e. IL-10, TGF-β, PGE2, IDO, HLA-G, and HGF) in tumours. These factors govern T lymphocyte activation and NK cell activity, and also induce Treg, governing the immune process [ 28 – 32 ]. The T-cell activation leads to a phenomenon of CD95L (FASLG) and becomes activated CD95-sensitive T cells. CD95-mediated apoptosis contributes to peripheral tolerance mechanisms by autoreactive T lymphocytes in the periphery. Thus, CD95L is employed in the defence of T-cell homeostasis. Also, the endothelial FASLG response is correlated with intratumoral CD8 + cells. The FASLG-governed T lymphocyte infiltration in tumours particularly leads to control of tumour-reactive CD8 + cells, thereby forming a CD8-to-FOXP3 T cell ratio and governed tumour growth [ 8 ]. Comparison of CD8+, CD4+, CD25+, and CD25 − cells revealed anti-apoptotic genes of BCL2 and BCL-XL and also the FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP). Also, c-FLIP L and c-FLIP S, but not BCL2 or BCL XL, in Treg cells enhanced their sensitivity by endothelial FASL. Further, transduction of CD8+, CD4 + and CD25- T cells with either c-FLIP S or c-FLIP L increased resistance by FASLG-mediated apoptosis. Thus, c-FLIP initiates FASL break in T lymphocytes and promotes c-FLIP activity is good for FASL interference in Treg cells. Further, FLIPs interfere with the response of apoptosis directly at the alignment of death receptors, and the IAPs bind and inhibit caspase. However, FASL signalling marked an increased rate in CD8 + TILs and the ratio of CD3+, CD8+, CD4+, CD25+, and FoxP3 + T cells (CD8/Treg) [ 33 , 8 ]. CD95L is led by the oligomerization of receptors and the conscription of FADD, MORT-1, and caspase-8. The FASL/DISC made the adaptor molecule of FADD, caspases 8, and caspases 10 initiate the phenomenon of apoptosis. FASL-initiated clustering of FAS, FADD, and caspase-8, caspase-10 within the DISC reveals autoproteolytic processing of caspases by active proteases. These molecules form an amino acid region called the death-inducing signalling complex (DISC). Within the DISC, pro-caspase-8 in proximity undergoes autocatalytic cleavage to stabilize caspase-8 and its catalytic conformation. Caspase-8 is released from the DISC through the cytosol and activates effector caspases, including caspase-3 and caspase-7, to drive the extrinsic apoptosis pathway. Several pieces of evidence support that caspase-10 is homologous to caspase-8 and activated by CD95-DISC [ 3 , 8 ]. The dynamism of caspase-10 among DISC triggers a switch from CD95L- and caspase-8-dependent cellular death by DISC-mediated activation of NF-kB signalling and survival. Also, procaspase-8 is cleaved auto-catalytically and initiates effector caspase directly or through a mitochondrial pathway. The formation of upshot caspases by FASL builds on a magnification curve on the caspase-initiated split of the BID (pro-apoptotic) and discharge of mitochondrial pro-apoptotic factors to drive the caspase-9-mediated apoptosome. Effective caspase-9 raises caspase-3 and activates caspase-8 without the FAS or DISC, thereby initiating a feedback loop [ 3 ]. Active effector caspase cleaves cellular substrates, signalling molecules, and ICAD, leading to the characteristic features of apoptosis. The CD95 response is initiated by pro-apoptotic and anti-apoptotic factors. However, major classes of molecules in the novel Bcl-2 family reveal cellular death at the mitochondrial level. The components in the defensive Bcl-2 family are divided into pro-apoptotic and anti-apoptotic molecules. Further, NFAT is dephosphorylated and bound to the promoter to induce CD95L [ 3 , 6 , 8 , 34 – 39 ]. In fibroblasts, CAFs confer the anti-tumour and T-lymphocyte responses by antigen-presenting cells. This mechanism drives the fibroblast process and cross-presenting the antigen complex with MHC-1. Further, antigen-specific FASL, PD-1, and PD-L2 on T lymphocytes suggested that CAFs drive the dysfunction and death of tumour-specific T lymphocytes and enhance survival. Those findings suggested that the PD-L2 and FASL enrichment within stromal cells through a CAF-mediated mechanism reveals tumour-associated fibroblasts and illustrate a unique mechanism of T cells within tumours. However, the nucleotide analogue 5-FU induces CD95L by a p53-dependent mechanism in tumour cells [ 40 ]. Also, tumours survive by phosphati-dylinositol 3-kinase (PI3K)/AKT signalling pathway. They also engage with the SAPK called JUN-N-terminal kinase or JNK signalling in neoplastic growth. Also, SAPKs are components in the MAPK family and are regulated by AP-1, which leads to CD95L. MAPK, exemplified by the redox state of cells, leads to the activation of AP-1 to induce CD95L. Further, SAPKs regulate inflammatory cytokines, such as TNF-α and IL-1β, in response to NF-κB. Also, the oxidative stress is triggered by the formation of ROS and glutathione in a response of CD95L [ 11 ]. These mechanisms also depend on ubiquitin-binding proteins, TAB2, TAB3, and TAK1. In such a phenomenon, the polyubiquitin scaffold recruits the inhibitor of nuclear factor-κB (IkB) kinase, containing IKK-alpha, IKK-beta, and IKK-gamma, in response to NF-kB. The IKK complex folds to the polyubiquitin scaffold through the ubiquitin-binding domain in NEMO [ 41 – 44 ]. Thus, epithelial cell-specific IKBKG (NEMO) encodes IKK, an inhibitor of NF-κB kinase, LPS-Rs, and lipopolysaccharide, which acts through Toll-like receptor 4, NF-κB, and TNF signalling. This link recruits TAB-TAK1, and further IKK complex allows TAK1 to phosphorylate IKK-beta [ 45 – 47 ]. The activated IKK complex stimulates NF-kB nuclear translocation and induces NF-kB-dependent transcription. In parallel, activated TAK1 triggers the MAPKKs to activate the JNK and p38 signalling. This combined function of NF-kB, JNK, and p38 promotes inflammation, immune responses (innate and adaptive) and cell survival [ 48 ]. So, molecular mechanisms behind cellular transformation are limited and need to be conserved in evolution. Therefore, the study is led by an analysis of the FASL gene and its family during cellular adaptation and survival. Concluding remarks The emerging paradigm supported by angiogenesis and immune suppression is linked with the biological phenomenon of tumour growth. Tumour-existing mechanisms are required to control inflammation and promote tissue rehabilitation during infection or wound healing, and the implementation of this program sustains tumour growth and promotes immunological tolerance. The study outcome focused on angiogenic tumour endothelium as a physical barrier, preventing T cell extravasations and being effective for anti-tumour immunity. Thus, the tumour endothelium and active immune regulators are controlled by the T cell function. However, FASL is a mediator of immune privilege, which is controversial, particularly in the context of cancer biology. Molecular alteration and cellular functions substantially affect the biological activity of FASL. This hypothesis demonstrated that the angiogenic growth factors induced by FASL response on the tumour endothelium, uniquely promote an immunosuppressive and tolerogenic phenomenon. Hence, the study outcome leads to FASLG-initiated mechanisms of immune tolerance and survival. Methods Target sequence and database The objective sequence is retrieved from various specific databases such as Universal Protein Resource (https://www.uniprot.org) (Morgat A et al. 2019), National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) (Eric W Sayers et al. 2019), European Molecular Biology Laboratory (https://www.embl.org), (W Baker et al. 2000), GenBank (https://www.ncbi.nlm.nih.gov/genbank) (EW Sayers et al. 2020), Kyoto Encyclopedia of Genes and Genomes (https://www.genome.jp/kegg) (Hiroyuki Ogata et al. 1999), and DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index-e.html) (T Okido et al. 2022). The web-based application of SMART (https://smart.embl.de/) (J Schultz et al. 1998) examines the particular peptide residues in objective sequence (query or suspected sequence). The SWISS-MODEL database (https://swissmodel.expasy.org/) retrieves for prediction of amino acids (peptides) structure. The above database is a bioinformatics web server for comparative modelling of the structure of protein molecules. This database generates a 3D structure utilized in effective research applications. The Swiss model is a regularly revised database of remodelling of organism proteome for biological research (T Schwede et al. 2003). Pfam (http://pfam.xfam.org) performs for particular protein family information. Also, PROCHECK (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK) web base tool performs for stereochemical quality of amino acids (peptide) structure (Laskowski R A et al. 1993). Genome sequence The organism’s genome sequences are downloaded from various specialized databases: 1. Ensembl Genomes (https://ensemblgenomes.org) (Kevin L Howe et al. 2020) and 2. NCBI (https://www.ncbi.nlm.nih.gov) (Eric W Sayers et al. 2019). Organisms 1. Homo sapiens : Genome assembly: GRCh38.p13 (GCA_000001405.28) 2. Mus musculus : Genome assembly: GRCm39 (GCA_000001635.9) Standalone Tools The HMMER (http://hmmer.org) software packages are rendered by MSA methods for the target residue as a contour search with parameters: 1.0e-3 in the genomes. However, HMMER is a mathematical and statistical algorithm assembled by a multiple sequence alignment (MSA) of the objective residue for profile search (SR Eddy. 2020). This implemented probabilistic model is well-known as an HMM (A Degirmenci. 2014). The standalone BLAST2 is implemented for homolog genes in organisms' genomes (Zhang J et al. 1997). Gene Annotation Gene ontology annotation is a method of functionally analyzing the genes in the genome across species for biological variance. So, the Omics box initializes using parameters 1.0e-3 for GO (gene ontology) annotation. Omics box (https://www.biobam.com/omicsbox) is a computational and bioinformatics application for high-throughput GO annotation of particular sequences in organisms (A Conesa et al. 2005). The functional property of genes rectified via gene ontology annotation is a popular tool for practical work (Ashburner et al. 2000). Domain Sequence domain analysis is a core component for the upgradation of conserved residue in two different organisms’ genomes. So, MSA methods are performed using a web-based application of MultAlin (http://multalin.toulouse.inra.fr/multalin) for analysis of the conserved region in sequences between both organisms. The MSA method calculates the specific pair of the homologous sequences and steak them up. Then observe the identical, differences, and similarities in sequences. The highest hits sequences are applied for MSA to upgrade the sustain domain (M Chatzou et al. 2016) (C Mitchell. 1993). Motifs The motif-based sequence analysis tools retrieve for resolution of sequence-specific motifs. MEME suite (https://meme-suite.org/meme) is a bioinformatics and computational web-based tool to analyze and validate particular motifs in the sequences (Timothy L et al. 2015). Phylogeny An analysis of phylogeny in genomes is necessary to explore the molecular Darwinism link between genes in both organisms. Therefore, MEGAX practice for constructing an evolutionary tree using Neighbor-Joining Methods (N Saitou et al. 1987) (Sudhir Kumar et al. 2018). Gene expression The gene expression analysis may be carried out by GENEVESTIGATOR (https://genevestigator.com). The GENEVESTIGATOR is an excessive search engine for gene expression of different organisms. That application is performed to determine and validate particular targets. Chromosome location The chromosomal location is retrieved through a gene card web-based application. The gene card (https://www.genecards.org) is a database of an organism's genes that provides knowledge on all recognized and predicted genes. The above database is updated and available for biomedical research (M Safran et al. 2010). Gene network The gene regulatory matrix reveals a molecular interaction in the cellular process to dominate the volume of mRNA or proteins. Some proteins act to activate genes as the TFs bind to the promoter area and initiate the response of different proteins known as regulatory cascades. STRING database retrieves for prediction of protein-protein interaction. STRING database (https://string-db.org) includes various resources like experimental data and computational prediction of nucleic acids and proteins (T Schlitt et al. 2003) (D Szklarczyk et al. 2017). Pathways The pathways analysis (PA) is generalized as functional enhancement analysis. So, PA is a widely accessible application for Biological Sciences. PA application helps in the examination of the biological preface of personalized genes to design and develop therapies. The KEGG (https://www.genome.jp/kegg) repository is accessible to retrieve and intellect the high-level function and utility of biological molecules like genes and proteins signal in a cellular process (Minoru Kanehisa. 2002). Abbreviations TNF Tumor necrosis factor TNFS Tumor necrosis factor superfamily TNFRS Tumor necrosis factor receptor superfamily NGF Nerve Growth Factor FASLG Fas ligand DNA Deoxyribonucleic acid BLAST Basic Local Alignment Search Tools HMM Hidden Markov Model GO Gene Ontology SD Standard Deviation MSA Multiple Sequence Alignment TNF Tumor necrosis factor TCR T cell receptor KEGG Kyoto Encyclopedia of Genes and Genomes UniProt Universal Protein Resource NCBI National Center for Biotechnology Information EMBL European Molecular Biology Laboratory DDBJ DNA Data Bank of Japan SMART Simple Modular Architecture Research Tool STRING Search Tool for the Retrieval of Interacting Genes/Proteins mRNA Messenger RNA RNA Ribonucleic acid NJM Neighbor-Joining Methods MEGA Molecular Evolutionary Genetics Analysis MEME Multiple Expectation maximizations for Motif Declarations Consent for Publication The work furnished in this paper is original and communicated by the correspondent placed in the manuscript. The author disclosed that the documents are not concerned elsewhere and have not been received for evaluation by other journals. Ethical approval: The study contains an in-silico analysis of the mammalian genome for upgradation and validation of particular genes in different organisms. Clinical trials: No applicable Availability of data and material: The data and samples may be available on request or demand. Conflict of interests: The author declared that the work has no conflict of interest. Funding: The author did not avail financial assistance from any source in undertaking the present study. Author Contribution This research paper was written by the sole author. SC proposed the idea, experimented, analyzed the data, and prepared the manuscript. Author information The author is a PhD research scholar at the Har Gobind Khorana School of Life Sciences, Department of Biotechnology, Assam University, Silchar-788011, Assam, India Acknowledgement: The author is grateful to Assam University, Silchar, Assam, India, for providing the lab facilities in carrying out this research work. References Shear MJP. Chemical Treatment of Tumors. IX. Reactions of Mice with Primary Subcutaneous Tumors to Injection of a Hemorrhage-Producing Bacterial Polysaccharide1. J Natl Cancer Inst. 1944;4:461–76. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci USA. 1975;3666–70. 10.1073/pnas.72.9.3666 . Dostert C, Grusdat M, Letellier E, Brenner D, THE TNF FAMILY OF LIGANDS AND RECEPTORS. COMMUNICATION MODULES IN THE IMMUNE SYSTEM AND BEYOND. Physiol Rev. 2019;99:115–60. Tracey KJ, Lowry SF, Cerami A. 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Endothelin B receptor mediates the endothelial barrier to T cell homing to tumors and disables immune therapy. Nat Med. 2008;14:28–36. Bouzin C, Brouet A, De Vriese J, Dewever J, Feron O. Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy. J Immunol. 2007;178:1505–11. Griffioen AW, Damen CA, Martinotti S, Blijham GH, Groenewegen G. Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic factors. Cancer Res. 1996;56:1111–7. Shetty S, et al. Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium. J Immunol. 2011;186:4147–55. Shrimali RK, et al. Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer. Cancer Res. 2010;70:6171–80. Basu GD, et al. Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: role of IDO. J Immunol. 2006;177:2391–402. Strasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in the immune system. Immunity. 2009;30:180–92. Yu JS, et al. Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium. J Neurooncol. 2003;64:55–61. Bajou K, et al. Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis. Cancer Cell. 2008;14:324–34. Sata M, Luo Z, Walsh K. Fas ligand overexpression on allograft endothelium inhibits inflammatory cell infiltration and transplant-associated intimal hyperplasia. J Immunol. 2001;166:6964–71. Yang J, et al. Endothelial cell overexpression of fas ligand attenuates ischemia-reperfusion injury in the heart. J Biol Chem. 2003;278:15185–91. Strauss L, Bergmann C, Whiteside TL. Human circulating CD4 + CD25highFoxp3 + regulatory T cells kill autologous CD8 + but not CD4 + responder cells by Fas-mediated apoptosis. J Immunol. 2009;182:1469–80. Krammer PH. CD95’s deadly mission in the immune system. Nature. 2000;407:789–95. Schmitz I, Kirchhoff S, Krammer PH. (2000) Regulation of death receptor-mediated apoptosis pathways. Int J Biochem Cell Biol. 2000; 32:1123–1136. Martinou JC, Green DR. (2001) Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol. 2001; 2:63–67. Zamzami N, Kroemer G. (2001) The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol. 2001; 2:67–71. Krueger A, Baumann S, Krammer PH, Kirchhoff S. FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol Cell Biol. 2001;21:8247–54. Deveraux QL, Reed JC. IAP family proteins–suppressors of apoptosis. Genes Dev. 1999;13:239–52. Krammer PH. The tumor strikes back. Cell Death Differ. 1997;4:362–4. Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ. Activation of IKK by TNFalpha requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell. 2006;22:245–57. Haas TL, Emmerich CH, Gerlach B, Schmukle AC, Cordier SM, Rieser E, Feltham R, Vince J, Warnken U, Wenger T, Koschny R, Komander D, Silke J, Walczak H. Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction. Mol Cell. 2006;36:831–44. Tokunaga F, Sakata S, Saeki Y, Satomi Y, Kirisako T, Kamei K, Nakagawa T, Kato M, Murata S, Yamaoka S, Yamamoto M, Akira S, Takao T, Tanaka K, Iwai K. Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation. Nat Cell Biol. 2009;11:123–32. Wu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. Nat Cell Biol. 2006;8:398–406. Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitindependent kinase of MKK and IKK. Nature. 2001;412:346–51. Rahighi S, Ikeda F, Kawasaki M, Akutsu M, Suzuki N, Kato R, Kensche T, Uejima T, Bloor S, Komander D, Randow F, Wakatsuki S, Dikic I. Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell. 2009;136:1098–109. Kanayama A, Seth RB, Sun L, Ea CK, Hong M, Shaito A, Chiu YH, Deng L, Chen ZJ. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 2004;15:535–48. Brenner D, Blaser H, Mak TW. Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol. 2015;15:362–74. Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Choudhury","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYDACCQYGZhBtwMDA+ABI8/ARq0UCqIXZAKSFjRQtbBIgAYJa5Gc3H5MuqLlXZ85++Fnl1xw7GTYG5oePbuDRYnDnWJr0jGPFEpY9aWa3ZbclAx3GZmycg0+LRI6ZNA9bgoTBgRy225LbmIFaeNik8WmRnwHS8g+o5fwbtmLJbfWEtTDcAGrhbQNquZHDxvhx22HCWgxupCVb8/YlSG648cxYmnHbcR42ZgJ+kZ+RfPA2z7cEfoPzyQ8//txWbc/P3vzwMV6HIQNmHjBJrHIQYPxBiupRMApGwSgYMQAA1CM/3v6aLJ8AAAAASUVORK5CYII=","orcid":"","institution":"Assam University","correspondingAuthor":true,"prefix":"","firstName":"Shouhartha","middleName":"","lastName":"Choudhury","suffix":""}],"badges":[],"createdAt":"2025-05-03 07:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6582476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6582476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83566468,"identity":"2ffd839e-7a75-4eec-b6f2-949a5ebe0467","added_by":"auto","created_at":"2025-05-28 15:06:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":868625,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.doc1.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/1d15feff436ea7daea7b677e.png"},{"id":83566469,"identity":"8cf710a4-b0d0-41d5-a022-da4cd97721dc","added_by":"auto","created_at":"2025-05-28 15:06:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33755,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for 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legend\u003c/p\u003e","description":"","filename":"Figure1.doc4.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/ec1854e64ea3424ccd667be6.png"},{"id":83567021,"identity":"74ef1043-e649-4f82-8b60-14ef858c29a0","added_by":"auto","created_at":"2025-05-28 15:14:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":72858,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.doc5.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/ee2313c683a56aa0be53b0d9.png"},{"id":83566474,"identity":"d2316b00-91a5-4f83-a39d-f36ff7f0294e","added_by":"auto","created_at":"2025-05-28 15:06:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":18081,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/725c6ae0aabd8831ac850f0b.png"},{"id":83566476,"identity":"82d9101b-b3cf-4732-80e1-b2006a93e68d","added_by":"auto","created_at":"2025-05-28 15:06:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":838064,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.doc7.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/f3a8fe99eeb11041da388755.png"},{"id":83567025,"identity":"5ff83cc5-3168-4336-b6d2-dbfd22e89a93","added_by":"auto","created_at":"2025-05-28 15:14:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":237714,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Figure1.doc8.png","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/47630c1d7873381321bf7076.png"},{"id":104782772,"identity":"ac1c53a7-09b8-4c18-a087-7dc542796b26","added_by":"auto","created_at":"2026-03-17 07:57:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3480247,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/3b5ee682-37cb-4d31-9e45-fc0eec74cbe3.pdf"},{"id":83566465,"identity":"269296c3-e55f-4921-add5-8e8dea6fd9b7","added_by":"auto","created_at":"2025-05-28 15:06:14","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11600,"visible":true,"origin":"","legend":"","description":"","filename":"Table.doc","url":"https://assets-eu.researchsquare.com/files/rs-6582476/v1/1f4e80cf7fd87a10c42ec912.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"In-silico analysis of FASLG gene resistance in Cancer Immunotherapy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the 19th century, William Coley proved that the tumours in some sarcoma patients contracted infections were triggered to necrotize. Further, it was discovered that lipopolysaccharides (LPS) are responsible for initiating the tumor-necrotizing activity. However, the biochemically and genetically described factor in serum provokes tumours to regulate molecules was proposed as a “tumour necrotizing factor” in 1975. This molecule, later called “tumour necrosis factor”, is a created component of a superfamily of closely related proteins [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e–\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Several reviews and data suggested that the classical TNF superfamily components are assumed through the plasma membrane via proteolytic cleavage (proteolysis) and represent a cytokine. Also, the fundamental nature of the superior TNF superfamily folds with the TNFR superfamily as a cytokine by the cysteine-rich residue. The TNFSF/TNFRSF ultimately includes 19 ligands \u0026amp; 29 receptors, revealing essential phenomena in the cellular process. So, the TNF superfamily is identified based on its gene sequence and functions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TNF-alpha and TNF-beta are first observed at the peptide level before isolation of their cDNA. Also, other components in the TNF superfamily were identified by cDNA sequence. The fundamental TNF superfamily includes TNF-alpha, TNF-beta, FASL, LT-beta, CD40L, CD27L, CD30L, 4-1BBL, RANKL, TRAIL, LIGHT, TWEAK, OX40L, APRIL, BAFF, GITRL, EDA-A1, EDA-A2, and VEGI. These members are binding to the 29 multiple TNF receptors. However, the terminology of the major TNF superfamily has ligands and receptors that are generalized by the International Commission. The TNF ligands are observed by the antigen-presenting cells, like dendritic cells, B lymphocytes and macrophages in the immune process. Also, the TNF ligands are standardized by NK cells, T lymphocytes, eosinophils, basophils, mast cells, epithelial cells, endothelial cells, and muscle cells. The signals initiated by TNFSF/TNFRSF are variable for homeostasis and growth in organisms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Also, TNFSF/TNFRSF components govern cellular adaptation, survival, and apoptosis, and their critical functions sustain immunity. The innate and adaptive immunity governed by TNFSF/TNFRSF components are valuable for the harmony of variant mechanisms leading to co-stimulation/co-inhibition. Malfunctions of the TNFSF/TNFRSF signalling are involved towards autoimmune and inflammatory disorders and underscore the magnitude of gene regulation. So, the concept of the TNFSF/TNFRSF activities led to decreased chronic inflammation and promoted anti-tumour immunity. Thus, the significant mechanisms underscore the molecular signalling that mediates the design and development of efficient anti-inflammatory and anti-cancer therapies [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e–\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Further concept of the TNF/NGF family reveals cognate receptors control immune functions, tissue homeostasis, and embryonic growth. So, the TNF/NGF family contributes to the decision of lymphocytes in the thymus. TNFR1, TNFR2, OX40, CD27, DR3, CD30, HVEM, 4-1BB, BAFF, TACI, and further transmembrane activators provide signals in antigen-stimulated T cells. Those receptors initiate stimulatory signals to enhance lymphocyte maturation and survival. These diverse natures are ordered by shared properties of family components by interactions of various factors with non-family ligands and co-receptors. The spectra are further prolonged by the phenomenon of ligands and receptors towards membrane-soluble by ensuring soluble formation to the extracellular matrix via signalling through intracellular domains [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. So, the major TNF superfamily is shaped by a homology domain that forms trimers. The classical tumour necrosis factor superfamily is a peptide family separated into two membrane polypeptides in the mammalian genome. The peptides in the classical TNF superfamily are conserved and located in the beta-sheet. The homology residue of the supreme TNF superfamily appears through molecular checkpoints and controls cellular functions (i.e. differentiation, proliferation, invasion, angiogenesis, embryogenesis, immune response, inflammation, and cellular death) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. So, the defensive TNF superfamily appears in immunologic, neurologic, myologic, pulmonary, and metabolic diseases. Concerning cancer therapy, studies in the late 1980s explored how cancer cell mortality was affected when treated through TNF, and the TNF provision of cohorts led to an inflammatory shock syndrome [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. TNF is a dynamic proinflammatory molecule, and its blocking activity has proved to be efficacious in decreasing the inflammation linked with autoimmunity. Immune-initiated mechanisms of tumours include antigen presentation and apoptosis resistance by active strategies and the production of immunosuppressive paracrine factors [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, CD95-induced selection of B cells is required for the homeostasis of both T and B cells. Studies of DC-specific CD95-deficient showed that CD95 plays a key role in DC homeostasis by controlling the immunogenicity of self-antigen presentation. Furthermore, CD95-mediated elimination of autoreactive T cells prevents the autoimmunity. So, the phenotypes observed by the lacking CD95L in T or B cells in organism. However, loss of CD95L specifically in CD4 + and CD8 + T cells did not affect the concentration of the T-cell components during illness. These results led to CD95L in T cells and homeostasis in response to acute stimuli, but are required to prevent autoimmunity mediated by chronically activated T cells. So, the inactivation of CD95 in T cells was accompanied by lymphopenia, because CD95L levels on activated T cells led to the apoptosis of peripheral lymphocytes.Thus, many tumours proved to reveal the apoptosis-inducing cell surface molecule called CD178 (CD95L, FASL, and APO-1L). The CD95L response can govern the maturation of killer T cells in an antigen-specific fashion. Also, immune reactions are prevented by major histocompatibility complex (MHC) and secretion of immunosuppressive cytokines. These mechanisms facilitate cancer growth in the immune process and have proven to be an immunotherapy [\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e–\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Immune therapy depends on several molecular functions and mechanisms that may promote tumour growth through the immune process. The immunologic theorem proposed that cancer immunotherapy builds on the molecular immune checkpoints that control T-cell activation. Those molecular mechanisms foster therapeutic applications in immuno-oncology [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, the study suggested an in-silico analysis of the FASLG gene from the TNF superfamily in mammalian genomes. The observation of TNF domain-mediated genes in the TNF superfamily is mandatory to explore the molecular function and mechanisms associated with T-cell immunity. So, perform bioinformatics and computational techniques to examine the TNF domain-initiated genes in the powerful TNF superfamily in two mammalian genomes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eStructural analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe primary sequence determined the concentration of nucleotides and peptides in the FASLG gene in \u003cem\u003eHomo sapiens\u003c/em\u003e.\u0026nbsp;Also, the sequence arranged by 846 nucleotides and 281 peptides among 137 peptides binds to DNA (\u003cstrong\u003eTable 1: Primary sequence of FASLG gene\u003c/strong\u003e). The nature of the FASLG is stable and forms homotrimers. Also, each promoter contains two beta-sheets and a conserved proline-rich residue employed in major intracellular transport and signalling (\u003cstrong\u003eFig. 1: 3D structure of FASLG\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome-wide analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome assembly determined the two model organisms of \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e. Further, the genome-wide examination of both organisms proposed the HMMER algorithm and obtained 47 and 15 TNF domains in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e, respectively (\u003cstrong\u003eTable 2: Summary of the (a) TNF domain (b) Homologs, and (c) FASLG\u003c/strong\u003e). Standalone BLAST2 outcome represents 43 and 14 homologs of FASLG genes in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e, respectively (\u003cstrong\u003eTable 2: Summary of the (a) TNF domain (b) Homologs, and (c) FASLG\u003c/strong\u003e). The gene ontology (GO) annotation confirmed the sequence accuracy of the TNF domain-mediated FASLG gene in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e (\u003cstrong\u003eTable 3: Summary of the genes in the TNF superfamily\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDomain, motifs, and phylogeny analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe highest hits of the CD95L (FASLG) were listed from both organisms for sequence alignment. The MSA results demonstrated a conserved TNF domain in FASLG (CD178) genes in both organisms. The ratio of consensus (50%) confirmed the extended TNF domain (\u003cstrong\u003eFig. 2:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eConserved TNF domain in FASLG gene\u003c/strong\u003e) and its specific motifs (\u003cstrong\u003eFig. 3:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSequence-specific motif\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in FASLG genes\u003c/strong\u003e) in the CD178 (CD95L). So, the observation concluded that the TNF domain is conserved in evolution. The phylogenetic tree suggested that the molecular evolutionary link of the TNF domain-mediated genes in-between \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e (\u003cstrong\u003eFig. 4: Molecular evolutionary link between FASLG and TNF-domain mediated genes in genomes\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosome location, gene network, gene expression, and pathway analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther, five categories of human mRNA explore one measure of the FASLG expression medium (log2 scale: 0.1) in the neoplasms of the eye, brain, central nervous system, and glioblastoma (\u003cstrong\u003eFig. 5:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExpression analysis of FASLG gene in human\u003c/strong\u003e). A chromosome location study confirmed that the FASLG gene is located at band 1q24.3. Started at 172,659,103 bp and stopped at 172,666,876 bp in humans (\u003cstrong\u003eFig. 6:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFASLG gene located at Chromosome 1 in human\u003c/strong\u003e). The gene network study determined that the\u0026nbsp;CD178 interacts with other molecules such as CASP10, CASP8, FOXO3, TNFRSF6B, TNFRSF1A, FAS, FADD, CFLAR, BID, and ENSG00000026036. Those molecular interactions govern the outcome of the CD95L in the cellular process (\u003cstrong\u003eFig. 6:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFASLG gene interacts with other TFs in cellular process\u003c/strong\u003e). Finally, the pathway study concluded the FASLG signalling in cellular death (\u003cstrong\u003eFig. 8:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFASLG signalling in cellular death\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe defensive immune system promises to be a key component of optimal cancer therapy. Effective strategies elicit immune replication and control tumours by enabling tumour-reactive T lymphocytes to infiltrate tumours. Cancer has dynamic levels of intra-tumoral T lymphocytes that significantly enhance survival across multiple tumours [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. So, T-cell aggression is essential for anti-tumour immunity and tumour mortality. Tumours employed complex biological phenomena linked with immune evasion and angiogenesis. Also, tumour angiogenesis is coordinated with the depth of T cell-mediated tumour rejection. So, the angiogenesis is perturbed by the conflict of the tumour endothelium. The tumour endothelium imposes substantial limits on T lymphocyte penetration, leading to tumour endothelial blockage. Thus, successful immunotherapy is based on molecular strategies to control the tumour endothelium. Consequently, the tumour endothelial barrier is coordinated by endothelial-T cells and governs T lymphocyte trafficking [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e–\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, a classical mechanism of dynamic pro-angiogenic growth factor of the VEGF-A governs endothelial-T cells by releasing VCAM-1 and intercellular resistance molecules (i.e. ICAM-1, ICAM-2, ICAM-3, ABCC1, and ABCB1) in endothelial cells. In contrast, the ABCDS (endothelin B receptor or ETBR) is linked with vascular concern and further controls T cell commitment to the endothelium. The response of VEGF-A/ETBR increases the rate of T-lymphocyte infiltration in tumours and promotes immune therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e–\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Also, VEGF-A with antibody (G6-31) and cyclooxygenase enzymes with acetylsalicylic acid (ASA) were effective in inducing tumour-infiltrating CD8 + T cells. The frequency of CD8 + cells in tumours was coordinated with the ratio of FASLG-positive vessels. Immune-initiated mechanisms were induced by FASL through T cell receptors in tumours and treated by VEGF-A-specific antibody and acetylsalicylic acid. However, granzyme-B (GrB) and interferon-γ (IFN-γ) respond in tumours treated by VEGF-A-specific antibodies and acetylsalicylic acid. Thus, ASA and VEGF-A-specific antibodies are treated by endothelial FASL, enabling CD8 + T lymphocyte infiltration in tumours. So, the IFN-γ and granzyme-B were linked with a ratio across tumours and treated by VEGF-A specific antibody and acetylsalicylic acid, suggesting cytotoxic T lymphocyte-initiated tumour suppression [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The mobilization of anti-tumour immunity, and lack of CD8 + cells, but not CD4 + cells, governs the ability of the VEGF-A-specific antibody and acetylsalicylic acid combination to inhibit tumour growth. Interestingly, CD4 + cells eliminated tumours and enhanced anti-tumour immunity. Numerous mechanisms are forwarded by the endothelium, revealing a critical function through T-cell groups [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the FASLG is a conventional homeostatic mediator of T cell and apoptosis and manifests in a tumour endothelium. FASLG is observed by vasculature and the penetration of pro-angiogenic and immunosuppressive paracrine factors (i.e. IL-10, TGF-β, PGE2, IDO, HLA-G, and HGF) in tumours. These factors govern T lymphocyte activation and NK cell activity, and also induce Treg, governing the immune process [\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The T-cell activation leads to a phenomenon of CD95L (FASLG) and becomes activated CD95-sensitive T cells. CD95-mediated apoptosis contributes to peripheral tolerance mechanisms by autoreactive T lymphocytes in the periphery. Thus, CD95L is employed in the defence of T-cell homeostasis. Also, the endothelial FASLG response is correlated with intratumoral CD8 + cells. The FASLG-governed T lymphocyte infiltration in tumours particularly leads to control of tumour-reactive CD8 + cells, thereby forming a CD8-to-FOXP3 T cell ratio and governed tumour growth [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Comparison of CD8+, CD4+, CD25+, and CD25 − cells revealed anti-apoptotic genes of BCL2 and BCL-XL and also the FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP). Also, c-FLIP L and c-FLIP S, but not BCL2 or BCL XL, in Treg cells enhanced their sensitivity by endothelial FASL. Further, transduction of CD8+, CD4 + and CD25- T cells with either c-FLIP S or c-FLIP L increased resistance by FASLG-mediated apoptosis. Thus, c-FLIP initiates FASL break in T lymphocytes and promotes c-FLIP activity is good for FASL interference in Treg cells. Further, FLIPs interfere with the response of apoptosis directly at the alignment of death receptors, and the IAPs bind and inhibit caspase. However, FASL signalling marked an increased rate in CD8 + TILs and the ratio of CD3+, CD8+, CD4+, CD25+, and FoxP3 + T cells (CD8/Treg) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. CD95L is led by the oligomerization of receptors and the conscription of FADD, MORT-1, and caspase-8. The FASL/DISC made the adaptor molecule of FADD, caspases 8, and caspases 10 initiate the phenomenon of apoptosis. FASL-initiated clustering of FAS, FADD, and caspase-8, caspase-10 within the DISC reveals autoproteolytic processing of caspases by active proteases. These molecules form an amino acid region called the death-inducing signalling complex (DISC). Within the DISC, pro-caspase-8 in proximity undergoes autocatalytic cleavage to stabilize caspase-8 and its catalytic conformation. Caspase-8 is released from the DISC through the cytosol and activates effector caspases, including caspase-3 and caspase-7, to drive the extrinsic apoptosis pathway. Several pieces of evidence support that caspase-10 is homologous to caspase-8 and activated by CD95-DISC [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The dynamism of caspase-10 among DISC triggers a switch from CD95L- and caspase-8-dependent cellular death by DISC-mediated activation of NF-kB signalling and survival. Also, procaspase-8 is cleaved auto-catalytically and initiates effector caspase directly or through a mitochondrial pathway. The formation of upshot caspases by FASL builds on a magnification curve on the caspase-initiated split of the BID (pro-apoptotic) and discharge of mitochondrial pro-apoptotic factors to drive the caspase-9-mediated apoptosome. Effective caspase-9 raises caspase-3 and activates caspase-8 without the FAS or DISC, thereby initiating a feedback loop [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Active effector caspase cleaves cellular substrates, signalling molecules, and ICAD, leading to the characteristic features of apoptosis. The CD95 response is initiated by pro-apoptotic and anti-apoptotic factors. However, major classes of molecules in the novel Bcl-2 family reveal cellular death at the mitochondrial level. The components in the defensive Bcl-2 family are divided into pro-apoptotic and anti-apoptotic molecules. Further, NFAT is dephosphorylated and bound to the promoter to induce CD95L [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e–\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In fibroblasts, CAFs confer the anti-tumour and T-lymphocyte responses by antigen-presenting cells. This mechanism drives the fibroblast process and cross-presenting the antigen complex with MHC-1. Further, antigen-specific FASL, PD-1, and PD-L2 on T lymphocytes suggested that CAFs drive the dysfunction and death of tumour-specific T lymphocytes and enhance survival. Those findings suggested that the PD-L2 and FASL enrichment within stromal cells through a CAF-mediated mechanism reveals tumour-associated fibroblasts and illustrate a unique mechanism of T cells within tumours. However, the nucleotide analogue 5-FU induces CD95L by a p53-dependent mechanism in tumour cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Also, tumours survive by phosphati-dylinositol 3-kinase (PI3K)/AKT signalling pathway. They also engage with the SAPK called JUN-N-terminal kinase or JNK signalling in neoplastic growth. Also, SAPKs are components in the MAPK family and are regulated by AP-1, which leads to CD95L. MAPK, exemplified by the redox state of cells, leads to the activation of AP-1 to induce CD95L. Further, SAPKs regulate inflammatory cytokines, such as TNF-α and IL-1β, in response to NF-κB. Also, the oxidative stress is triggered by the formation of ROS and glutathione in a response of CD95L [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These mechanisms also depend on ubiquitin-binding proteins, TAB2, TAB3, and TAK1. In such a phenomenon, the polyubiquitin scaffold recruits the inhibitor of nuclear factor-κB (IkB) kinase, containing IKK-alpha, IKK-beta, and IKK-gamma, in response to NF-kB. The IKK complex folds to the polyubiquitin scaffold through the ubiquitin-binding domain in NEMO [\u003cspan additionalcitationids=\"CR42 CR43\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e–\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, epithelial cell-specific IKBKG (NEMO) encodes IKK, an inhibitor of NF-κB kinase, LPS-Rs, and lipopolysaccharide, which acts through Toll-like receptor 4, NF-κB, and TNF signalling. This link recruits TAB-TAK1, and further IKK complex allows TAK1 to phosphorylate IKK-beta [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e–\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The activated IKK complex stimulates NF-kB nuclear translocation and induces NF-kB-dependent transcription. In parallel, activated TAK1 triggers the MAPKKs to activate the JNK and p38 signalling. This combined function of NF-kB, JNK, and p38 promotes inflammation, immune responses (innate and adaptive) and cell survival [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. So, molecular mechanisms behind cellular transformation are limited and need to be conserved in evolution. Therefore, the study is led by an analysis of the FASL gene and its family during cellular adaptation and survival.\u003c/p\u003e "},{"header":"Concluding remarks","content":"\u003cp\u003eThe emerging paradigm supported by angiogenesis and immune suppression is linked with the biological phenomenon of tumour growth. Tumour-existing mechanisms are required to control inflammation and promote tissue rehabilitation during infection or wound healing, and the implementation of this program sustains tumour growth and promotes immunological tolerance. The study outcome focused on angiogenic tumour endothelium as a physical barrier, preventing T cell extravasations and being effective for anti-tumour immunity. Thus, the tumour endothelium and active immune regulators are controlled by the T cell function. However, FASL is a mediator of immune privilege, which is controversial, particularly in the context of cancer biology. Molecular alteration and cellular functions substantially affect the biological activity of FASL. This hypothesis demonstrated that the angiogenic growth factors induced by FASL response on the tumour endothelium, uniquely promote an immunosuppressive and tolerogenic phenomenon. Hence, the study outcome leads to FASLG-initiated mechanisms of immune tolerance and survival.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eTarget sequence and database\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe objective sequence is retrieved from various specific databases such as\u0026nbsp;Universal Protein Resource (https://www.uniprot.org) (Morgat A et al. 2019), National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) (Eric W Sayers et al. 2019), European Molecular Biology Laboratory (https://www.embl.org), (W Baker et al. 2000), GenBank (https://www.ncbi.nlm.nih.gov/genbank) (EW Sayers et al. 2020), Kyoto Encyclopedia of Genes and Genomes (https://www.genome.jp/kegg) (Hiroyuki Ogata et al. 1999), and DNA Data Bank of Japan (https://www.ddbj.nig.ac.jp/index-e.html) (T Okido et al. 2022). The web-based application of SMART (https://smart.embl.de/) (J Schultz et al. 1998) examines the particular peptide residues in objective sequence (query or suspected sequence). The SWISS-MODEL database (https://swissmodel.expasy.org/) retrieves for prediction of amino acids (peptides) structure. The above database is a bioinformatics web server for comparative modelling of the structure of protein molecules. This database generates a 3D structure utilized in effective research applications. The Swiss model is a regularly revised database of remodelling of organism proteome for biological research (T Schwede et al. 2003). Pfam (http://pfam.xfam.org) performs for particular protein family information. Also, PROCHECK (https://www.ebi.ac.uk/thornton-srv/software/PROCHECK) web base tool performs for stereochemical quality of amino acids (peptide) structure (Laskowski R A et al. 1993).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome sequence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe organism\u0026rsquo;s genome sequences are downloaded from various specialized databases:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1. Ensembl Genomes (https://ensemblgenomes.org) (Kevin L Howe et al. 2020) and\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2. NCBI (https://www.ncbi.nlm.nih.gov)\u0026nbsp;(Eric W Sayers et al. 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOrganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 1. \u003cem\u003eHomo sapiens\u003c/em\u003e: Genome assembly: GRCh38.p13 (GCA_000001405.28)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 2. \u003cem\u003eMus musculus\u003c/em\u003e: Genome assembly: GRCm39 (GCA_000001635.9)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandalone Tools\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe HMMER (http://hmmer.org) software packages are rendered by MSA methods for the target residue as a contour search with parameters: 1.0e-3 in the genomes. However, HMMER is a mathematical and statistical algorithm assembled by a multiple sequence alignment (MSA) of the objective residue for profile search (SR Eddy. 2020). This implemented probabilistic model is well-known as an HMM (A Degirmenci. 2014). The standalone BLAST2 is implemented for homolog genes in organisms\u0026apos; genomes (Zhang J et al. 1997).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene Annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene ontology annotation is a method of functionally analyzing the genes in the genome across species for biological variance. So, the Omics box initializes using parameters 1.0e-3 for GO (gene ontology) annotation. Omics box (https://www.biobam.com/omicsbox) is a computational and bioinformatics application for high-throughput GO annotation of particular sequences in organisms (A Conesa et al.\u0026nbsp;2005). The functional property of genes rectified via gene ontology annotation is a popular tool for practical work (Ashburner et al. 2000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDomain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence domain analysis is a core component for the upgradation of conserved residue in two different organisms\u0026rsquo; genomes. So, MSA methods are performed using a web-based application of MultAlin (http://multalin.toulouse.inra.fr/multalin) for analysis of the conserved region in sequences between both organisms. The MSA method calculates the specific pair of the homologous sequences and steak them up. Then observe the identical, differences, and similarities in sequences. The highest hits sequences are applied for MSA to upgrade the sustain domain (M Chatzou et al. 2016) (C Mitchell. 1993).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMotifs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe motif-based sequence analysis tools retrieve for resolution of sequence-specific motifs. MEME suite (https://meme-suite.org/meme) is a bioinformatics and computational web-based tool to analyze and validate particular motifs in the sequences (Timothy L et al. 2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogeny\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn analysis of phylogeny in genomes is necessary to explore the molecular Darwinism link between genes in both organisms. Therefore, MEGAX practice for constructing an evolutionary tree using\u0026nbsp;Neighbor-Joining Methods\u0026nbsp;(N Saitou et al. 1987) (Sudhir Kumar et al. 2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene expression analysis may be carried out by GENEVESTIGATOR (https://genevestigator.com). The GENEVESTIGATOR is an excessive search engine for gene expression of different organisms. That application is performed to determine and validate particular targets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosome location\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromosomal location is retrieved through a gene card web-based application. The gene card (https://www.genecards.org) is a database of an organism\u0026apos;s genes that provides knowledge on all recognized and predicted genes. The above database is updated and available for biomedical research (M Safran et al. 2010).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene network\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene regulatory matrix reveals a molecular interaction in the cellular process to dominate the volume of mRNA or proteins. Some proteins act to activate genes as the TFs bind to the promoter area and initiate the response of different proteins known as regulatory cascades. STRING database retrieves for prediction of protein-protein interaction. STRING database (https://string-db.org) includes various resources like experimental data and computational prediction of nucleic acids and proteins (T Schlitt et al. 2003) (D Szklarczyk et al. 2017).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pathways analysis (PA) is generalized as functional enhancement analysis. So, PA is a widely accessible application for Biological Sciences. PA application helps in the examination of the biological preface of personalized genes to design and develop therapies. The KEGG (https://www.genome.jp/kegg) repository is accessible to retrieve and intellect the high-level function and utility of biological molecules like genes and proteins signal in a cellular process (Minoru Kanehisa. 2002).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNFS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor superfamily\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNFRS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor receptor superfamily\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNerve Growth Factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFASLG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFas ligand\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasic Local Alignment Search Tools\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHidden Markov Model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStandard Deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultiple Sequence Alignment\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT cell receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUniProt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUniversal Protein Resource\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMBL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEuropean Molecular Biology Laboratory\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDDBJ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDNA Data Bank of Japan\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMART\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimple Modular Architecture Research Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSTRING\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSearch Tool for the Retrieval of Interacting Genes/Proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMessenger RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRibonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNJM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeighbor-Joining Methods\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMolecular Evolutionary Genetics Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMEME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMultiple Expectation maximizations for Motif\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work furnished in this paper is original and communicated by the correspondent placed in the manuscript. The author disclosed that the documents are not concerned elsewhere and have not been received for evaluation by other journals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study contains an\u0026nbsp;in-silico\u0026nbsp;analysis of the mammalian genome for upgradation and validation of particular genes in different organisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trials:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and samples may be available on request or demand.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declared that the work has no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author did not avail financial assistance from any source in undertaking the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research paper was written by the sole author. SC proposed the idea, experimented, analyzed the data, and prepared the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author is a PhD research scholar at the Har Gobind Khorana School of Life Sciences, Department of Biotechnology, Assam University, Silchar-788011, Assam, India\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author is grateful to Assam University, Silchar, Assam, India, for providing the lab facilities in carrying out this research work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShear MJP. Chemical Treatment of Tumors. IX. Reactions of Mice with Primary Subcutaneous Tumors to Injection of a Hemorrhage-Producing Bacterial Polysaccharide1. 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J Immunol. 2007;178:1505\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriffioen AW, Damen CA, Martinotti S, Blijham GH, Groenewegen G. Endothelial intercellular adhesion molecule-1 expression is suppressed in human malignancies: the role of angiogenic factors. Cancer Res. 1996;56:1111\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShetty S, et al. Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium. J Immunol. 2011;186:4147\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShrimali RK, et al. Antiangiogenic agents can increase lymphocyte infiltration into tumor and enhance the effectiveness of adoptive immunotherapy of cancer. Cancer Res. 2010;70:6171\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasu GD, et al. Cyclooxygenase-2 inhibitor enhances the efficacy of a breast cancer vaccine: role of IDO. J Immunol. 2006;177:2391\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in the immune system. Immunity. 2009;30:180\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu JS, et al. Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium. J Neurooncol. 2003;64:55\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBajou K, et al. Plasminogen activator inhibitor-1 protects endothelial cells from FasL-mediated apoptosis. Cancer Cell. 2008;14:324\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSata M, Luo Z, Walsh K. Fas ligand overexpression on allograft endothelium inhibits inflammatory cell infiltration and transplant-associated intimal hyperplasia. J Immunol. 2001;166:6964\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, et al. Endothelial cell overexpression of fas ligand attenuates ischemia-reperfusion injury in the heart. J Biol Chem. 2003;278:15185\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStrauss L, Bergmann C, Whiteside TL. Human circulating CD4 + CD25highFoxp3 + regulatory T cells kill autologous CD8 + but not CD4 + responder cells by Fas-mediated apoptosis. J Immunol. 2009;182:1469\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrammer PH. CD95\u0026rsquo;s deadly mission in the immune system. Nature. 2000;407:789\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmitz I, Kirchhoff S, Krammer PH. (2000) Regulation of death receptor-mediated apoptosis pathways. Int J Biochem Cell Biol. 2000; 32:1123\u0026ndash;1136.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartinou JC, Green DR. (2001) Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol. 2001; 2:63\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZamzami N, Kroemer G. (2001) The mitochondrion in apoptosis: how Pandora\u0026rsquo;s box opens. Nat Rev Mol Cell Biol. 2001; 2:67\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrueger A, Baumann S, Krammer PH, Kirchhoff S. FLICE-inhibitory proteins: regulators of death receptor-mediated apoptosis. Mol Cell Biol. 2001;21:8247\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeveraux QL, Reed JC. IAP family proteins\u0026ndash;suppressors of apoptosis. Genes Dev. 1999;13:239\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrammer PH. The tumor strikes back. 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Nat Cell Biol. 2009;11:123\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu CJ, Conze DB, Li T, Srinivasula SM, Ashwell JD. Sensing of Lys 63-linked polyubiquitination by NEMO is a key event in NF-kappaB activation [corrected]. Nat Cell Biol. 2006;8:398\u0026ndash;406.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitindependent kinase of MKK and IKK. Nature. 2001;412:346\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahighi S, Ikeda F, Kawasaki M, Akutsu M, Suzuki N, Kato R, Kensche T, Uejima T, Bloor S, Komander D, Randow F, Wakatsuki S, Dikic I. Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation. Cell. 2009;136:1098\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanayama A, Seth RB, Sun L, Ea CK, Hong M, Shaito A, Chiu YH, Deng L, Chen ZJ. TAB2 and TAB3 activate the NF-kappaB pathway through binding to polyubiquitin chains. Mol Cell. 2004;15:535\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrenner D, Blaser H, Mak TW. Regulation of tumour necrosis factor signalling: live or let die. Nat Rev Immunol. 2015;15:362\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"[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":"FASLG, TNF Family, Cancer, Gene Therapy, Immunotherapy","lastPublishedDoi":"10.21203/rs.3.rs-6582476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6582476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe FASLG gene in the TNF superfamily binds with the receptor superfamily to induce apoptosis. FAS ligand and receptor interactions lead to a dominant nature in the immune process and control cellular death (apoptosis). The dynamism of the apoptotic-induced FASLG gene controls tumour-infiltrating lymphocytes and suppresses tumour responses called tumour counter-attacks. Intense evidence in the animal model illustrated that the FASLG (CD95L) gene functions govern T-cell revival and tumour exit. The justification of CD95L (CD178) controls tumours and induces inflammation. Those mechanisms forwarded the T-cell back into the tumour context by immune checkpoints restraining tumour growth. Also, the mechanisms suggested that CD178 in tumour cells contributes to immune escape.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eSo, the study proposed an in-silico analysis of the FASLG gene and its TNF family to pursue the molecular immunologic mechanisms linked with cancer and immunity in the model organisms. Hence, the study utilized bioinformatics and computational applications to learn about the TNF domain-mediated genes in two isolated organism\u0026rsquo;s genome. This application proposed a significant way to study particular genes in the genome.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA genome-wide observation suggested multiple hits of the TNF domains in the fundamental TNF superfamily in \u003cem\u003eHomo sapiens\u003c/em\u003e and \u003cem\u003eMus musculus\u003c/em\u003e. Further observation of the FASLG gene demonstrated the nucleotides, peptides, 3D structure, domain, motifs, phylogeny, gene expression, gene network, chromosome location, and signalling pathway in humans. The study remarked that the FASLG gene and its functional mechanisms are integrated with the immune process.\u003c/p\u003e\u003ch2\u003eConcluding remarks:\u003c/h2\u003e \u003cp\u003eThe documented data forward the FALSG gene and its molecular mechanisms during T-cell initiation in the tumour microenvironment. Those molecular mechanisms proposed T-cell suppression by immune checkpoints and control of tumour progression via anti-tumour immune response.\u003c/p\u003e","manuscriptTitle":"In-silico analysis of FASLG gene resistance in Cancer Immunotherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 15:06:09","doi":"10.21203/rs.3.rs-6582476/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":"1811a753-b2ef-4e53-885a-2dcd8e2fbdcf","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T09:29:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-28 15:06:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6582476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6582476","identity":"rs-6582476","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}