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bioRxiv2025

Characteristics predicting reduced penetrance variants in the high-risk cancer predisposition gene TP53

Disease-causing variants with penetrance that is lower than the average expected for a given gene complicate classification, even when using gene-specific guidelines. For TP53, a gene associated with some of the highest cancer risks, even reduced penetrance disease-predisposition variants remain clinically actionable. We conducted a review of ClinVar submissions to identify TP53 variants flagged as having reduced penetrance by genetic testing laboratories and analyzed functional, bioinformatic, frequency, and clinical features of these variants compared to standard pathogenic and benign variants. Our findings show that reported reduced penetrance TP53 variants are more likely to exhibit intermediate functional activity in multiple assays and are predicted as deleterious with bioinformatic tools, though with lower scores than pathogenic variants. These variants also have a higher population frequency than pathogenic variants, and heterozygotes tend to manifest disease later in life, suggesting a need for refined clinical criteria to better capture attenuated Li-Fraumeni syndrome phenotypes. Finally, by applying a random forest prediction model to all TP53 uncertain or conflicting variants in ClinVar, we identified additional variants with potential reduced penetrance.

genetics↗

bioRxiv2025

Differential Proteomic Analysis of DEN-Induced Hepatocellular Carcinoma in Male and Female Balb/c Mice Reveals Novel Gender Specific Markers

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of hepatic malignancy with a higher prevalence in males compared to females, however, the distinct underlying mechanisms contributing to this disparity remain poorly understood. MethodsIn this study, we aimed to investigate comparative proteome profiling of a diethylnitrosamine (DEN) induced HCC model in male and female Balb/c mice. We extracted proteins from liver tissue of DEN treated male and female mice and their corresponding controls and subjected them to mass spectrometry and subsequent bioinformatics analyses. ResultsWe identified 170 and 233 differentially expressed proteins (DEPs) in female and male mice, respectively. We identified chemical carcinogenesis and cortical cytoskeleton as the shared pathways between the two groups. In addition, we identified distinct signaling pathways in DEN-treated male and female mice. Female mice showed enrichment in oxidative phosphorylation, fatty acid biosynthesis, metabolism and degradation and cytochrome P450 clusters. In contrast, in male mice, these pathways were enriched in cholesterol metabolism, coagulation and mRNA processing. Further, we identified top ten genes ranked by highest maximal clique centrality, by protein-protein interaction analysis of differentially expressed proteins (DEPs) in both sexes. Of these hub genes, female mice showed upregulation of NDUFA8 and ATP5H and were associated with poor patient survival. On the other hand, In DEN-treated male mice upregulation of FGG, FGA, HPX and SERPINC1 were associated with poor survival. ConclusionIn conclusion, our research provides gender-specific proteomic signatures in DEN-induced HCC. The identification of proteins associated cholesterol metabolism and coagulation in males, and mitochondrial complex I proteins in females as prognostic markers suggests novel therapeutic targets that may inform gender-tailored treatment strategies for HCC. Simple SummaryHepatocellular carcinoma (HCC) is a common and deadly liver cancer that affects men more than women. To understand the biological reasons behind this difference, we developed a liver cancer model in male and female mice using a chemical called diethylnitrosamine (DEN). We then studied the proteins involved in tumor development using advanced techniques like mass spectrometry and bioinformatics. We found that different sets of proteins and biological pathways were active in males and females. In males, ribosomal and RNA-binding proteins were linked to worse survival, while in females, mitochondrial proteins were more important. These findings suggest that men and women may need different strategies for diagnosing and treating HCC, and they offer new gender-specific targets for future therapies.

cancer biology↗

bioRxiv2025

PIWIL2 downregulation in colon cancer promotes transposon activity and pro-tumorigenic phenotypes

Reactivation of transposable elements (TEs) in somatic tissues, particularly of LINE-1, is associated with disease by causing gene mutations and DNA damage. Previous work has shown that the PIWI pathway is crucial for TE suppression in the germline. However, the status and function of this pathway is not well characterized in differentiated somatic cells and there is lack of consensus on the role of the pathway in somatic tumorigenesis. To shed light on this conundrum, we examined the PIWI pathway in colon cancer through combining bioinformatic analyses and cell-based assays. Shifted Weighted Annotation Network (SWAN) analysis revealed that the pathway experiences significant allelic losses in colon cancer and that PIWIL2, the main catalytic component of the pathway responsible for TE silencing, experiences the highest percent deletions. PIWIL2 is downregulated in colon tumors of advanced stage, nodal metastasis, and in certain subtypes, correlating with poor survival, while it is also downregulated in ulcerative colitis, an inflammatory bowel disease that predisposes to colon cancer. PIWIL2 depletion in colon epithelial Caco2 cells leads to increased anchorage-independent growth, loss of transposon-targeting - non-canonical - piRNAs, increased LINE-1 levels and activity, and in DNA damage, altogether highlighting a tumor-suppressing role of PIWIL2 in the colon. SUMMARY STATEMENTThis study investigates the PIWI-piRNA pathway in colon cancer using a nuanced bioinformatic and cell-based approach, linking the downregulation of PIWIL2 to disease progression, transposable element activation and DNA damage.

cancer biology↗

bioRxiv2025

A curated dataset and web application for Integrative Analysis of Ancient DNA from Britain and Ireland

In recent years, genome-wide data from thousands of ancient human remains have been sequenced, offering unprecedented insights into past populations. This vast dataset of ancient human genomes and their associated metadata holds immense value for archaeologists, anthropologists, and biologists. However, extracting insights often requires advanced bioinformatics knowledge, creating barriers for non-experts. To address this challenge, we present a curated dataset and user-friendly web application for published genome-wide data from ancient Britain and Ireland, including new data produced by the COMMIOS Project. The platform integrates visualization tools, enabling researchers to explore genetic connections between ancient and modern populations without requiring bioinformatics expertise.

genetics↗

bioRxiv2025

Detection of astrocyte epigenetic memory in in vitro systems, experimental autoimmune encephalomyelitis and multiple sclerosis samples

We recently described astrocyte pro-inflammatory epigenetic memory based on multiple complementary in vivo and in vitro studies, and the analysis of multiple sclerosis samples. Based on bioinformatic analyses, ODea and Liddelow argued that the astrocyte epigenetic memory we described is the result of contamination with immune cells, particularly myeloid cells. We rebut ODea and Liddelow arguments as follows: (1) We show substantial purity of astrocytes analyzed in in vivo and in vitro systems; (2) We recapitulate astrocyte memory responses using five independent pure astrocyte in vitro systems, and show its dependency on the histone acetyl transferase p300; and (3) Using the Liddelow lab bioinformatic pipeline to implement purity and cell-quality criteria, we detect astrocyte epigenetic memory in five independent scRNA-seq experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) astrocyte datasets. These additional analyses and studies provide further support for the existence of astrocyte pro-inflammatory epigenetic memory.

neuroscience↗

bioRxiv2025

Astrocytic CREB regulates transcriptional, neuronal, and behavioral responses to cocaine

Drug addiction is characterized by neuronal adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. Emerging evidence increasingly indicates that astrocytes are also involved in nervous system disorders, including addiction, but the cocaine-induced astrocyte-specific transcriptome has not yet been investigated. We utilized whole cell sorting of astrocytes, RNA-sequencing, and bioinformatic approaches to characterize the astrocyte transcriptome in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, prolonged abstinence, and "relapse" in male mice. We found that astrocytes exhibit robust and contextually-specific transcriptional signatures that converge strongly with human cocaine use disorder. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator of cocaine-induced transcriptional regulation in NAc astrocytes, and CUT&RUN-sequencing mapped increased CREB binding across the astrocyte genome in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes, in combination with several measures of addiction-related behaviors including conditioned place preference and self-administration, revealed that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in astrocytic CREB activity or CPP found in females. Subsequent experiments identify potential molecular mechanisms of astrocytic CREBs influence through modulating astrocytic Ca2+ signaling in response to cocaine. Finally, we show that astrocytic CREB selectively modulates D1-type medium spiny neurons in NAc to control cocaine-related behaviors. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine and that CREB mediates cocaines effects on gene expression in astrocytes, with consequent effects on neuronal activity and rewarding responses to the drug.

neuroscience↗

bioRxiv2025

High-resolution cryo-EM analysis of the therapeutic Pseudomonas phage Pa223

Cryogenic electron microscopy (cryo-EM) analysis of bacteriophages is a valuable method for deciphering virus composition and conformational plasticity. In this study, we present a high-resolution structural atlas of the Pseudomonas virus Pa223, a phage from the Bruynoghevirus genus that has recently been used in clinical cocktails for treating cystic fibrosis and non-cystic fibrosis bronchiectasis, as well as for compassionate care. By combining bioinformatics, proteomics, cryo-EM single particle analysis, and localized reconstruction, we annotated and built atomic models for eight structural polypeptide chains that form the icosahedral capsid and noncontractile tail. We discovered that the Pa223 capsid is decorated by a spike protein that features a unique triple-{beta} helix fold with no structural homologs in the database. The Pa223 tail features six trimeric tail fibers extending upwards, similar to, but shorter than, those found in phage T7. Unlike T7, the Pa223 tail is extended by two head-to-tail adaptors and sealed by a trimeric tail needle, similar to P22-like phages. We identified a protein bound around the outer perimeter of the portal protein, positioned similarly to the ejection protein gp72, which was identified in the Pseudomonas phage DEV, a Litunavirus phage and member of the reclassified Schitoviridae family. This structural hint led us to identify the Pa223 ejection proteins gp53, gp54, and gp56, which bioinformatically resemble those of T7-like phages more closely than Schitoviridae. Thus, phage Pa223 contains diverse structural elements found in P22-like, T7-like, and Litunavirus phages, providing a framework for understanding the diversification and evolution of ejection proteins in Bruynogheviruses. HighlightsO_LIThe high-resolution structure of Bruynoghevirus Pa223 reveals hybrid structural features that are shared among P22-like, T7-like, and Litunavirus phages. C_LIO_LIThe Pa223 capsid is decorated with a trimeric spike asymmetrically bound at the icosahedral 3-fold axes. C_LIO_LIThe Pa223 tail features two quasi-equivalent conformations of the head-to-tail adaptor protein arranged into two coaxial rings. C_LIO_LIIdentification of the ejection protein gp54 through structural similarity to gp72 from the Litunavirus DEV. C_LIO_LIBioinformatic mapping of the Pa223 ejection proteins gp53 and gp56 validated through mass spectrometry analysis of infectious virions. C_LI

biochemistry↗

bioRxiv2025

Scalable single-cell metagenomic analysis with Bascet and Zorn

Single-cell metagenomic sequencing (scMetaG) can provide maximum-resolution insights into complex microbial communities. However, existing bioinformatic tools are not equipped to handle the massive amounts of data generated by novel high-throughput scMetaG methods. Here, we present a bioinformatic toolkit for complete, end-to-end scMetaG analysis: (i) Bascet, a command-line suite designed to scale to massive scMetaG datasets ([≥]1 million cells); (ii) Zorn, an R package/workflow manager that enables reproducible scMetaG data analysis, exploration, and visualization (http://zorn.henlab.org/). Enabled by recent advances in droplet microfluidics, we use Bascet and Zorn to develop and optimize a high-throughput scMetaG method on a ten-species mock community. To showcase their utility on a real-world sample, we use Bascet and Zorn to characterize a human saliva sample, generating single-amplified genomes (SAGs) from >10k prokaryotic cells. Overall, Bascet and Zorn enable reproducible scMetaG analysis, allowing users to query microbiomes at unprecedented resolution and scale.

microbiology↗

bioRxiv2025

Deciphering the Nrf2/ARE Mechanism: GanCaoXieXin Decoction Combats Oxidative Stress in Ulcerative Colitis Pathogenesis

BackgroundGanCaoXieXin (GCXX) decoction, a classic prescription, has shown clinical efficacy in treating ulcerative colitis (UC). However, its mechanism remains incompletely understood. ObjectiveThis study aims to explore how GCXX modulates the Nrf2/ARE signaling pathway to mitigate oxidative stress (OS)-induced damage and thereby ameliorate UC. Materials and methodsNetwork pharmacology and bioinformatics analyses identified key targets of GCXX in UC treatment. Ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry (UHPLC-MS/MS) analyzed GCXXs effective compounds. A 2.5% Dextran Sulfate Sodium Salt (DSS)-induced UC mouse model was used. Immunohistochemistry (IHC) assessed tight junction proteins. Enzyme-linked immunosorbent assay (ELISA) measured intestinal permeability and oxidative stress markers. Western blot (WB) analyzed Nrf2/ARE signaling proteins. In 800mol/L H2O2-induced oxidative stress (OS) state HT-29 cells, cell viability, apoptosis, oxidative stress indicators, and apoptosis-related proteins were evaluated. Immunofluorescence (IF) detected Nrf2/ARE signaling axis proteins. ResultsThe results of network pharmacology and bioinformatics analysis revealed that GCXX could intervene in UC by regulating OS-related pathways. GCXX contained antioxidant components like quercetin, berberine, and baicalin. In vivo, GCXX alleviated mucosal damage, reduced intestinal permeability, downregulated MDA, upregulated SOD, suppressed Keap1, and promoted tight junction and Nrf2/ARE pathway proteins. In vitro, GCXX increased cell survival, improved antioxidant capacity, reduced MDA and apoptosis in OS state cells. Immunofluorescence confirmed Nrf2/ARE pathway as crucial in GCXXs protective effects. ConclusionsGCXX elevates the expression level of Nrf2, HO-1 and NQO1, thus reducing entercell OS-induced damage, intestinal cell apoptosis, alleviating UC intestinal mucosal damaget.

pharmacology and toxicology↗

bioRxiv2025

Structural similarities reveal an expansive conotoxin family with a two-finger toxin fold

Venomous animals have evolved a diverse repertoire of toxins with considerable pharmaceutical potential. The rapid evolution of peptide toxins, such as the conotoxins produced by venomous marine cone snails, often complicates efforts to infer their evolutionary relationships based solely on sequence information. Structural bioinformatics, however, can provide robust support. Here, we first solve the NMR structure of a macro-conotoxin from the MLSML superfamily, Tx33.1, which is composed of 124 residues, including 12 cysteines. We then apply deep learning-based methods for structure prediction and comparison to identify structural similarities between this toxin and five additional, previously uncharacterized conotoxin superfamilies. Although only three of these superfamilies exhibit sequence homology, a combined approach incorporating structure prediction, structure comparison, and gene structure analysis supports the conclusion that all six superfamilies share a common evolutionary past. The Tx33.1 NMR structure displays similarity to the first two domains of Argos, a secretory protein from Drosophila melanogaster that comprises three domains, each harboring two short {beta}-stranded loops ("fingers"). Consequently, we propose the name "two-finger toxin (2FTX)" fold for this type of domain. Finally, using structure similarity searches, we identify a wide range of 2FTX proteins in protostomes, including non-venom-derived, secretory cone snail proteins. This study demonstrates how structural bioinformatics can be employed to uncover evolutionary relationships among rapidly evolving genes. It simultaneously identifies a large, previously unrecognized group of protostome 2FTX proteins, many of which exhibit close structural similarity to Argos and may perform a similar function in regulating EGFR signaling.

evolutionary biology↗

bioRxiv2025

High-throughput experimental validation of novel hairpin ribozymes

The small self-cleaving hairpin ribozyme has served as a model for RNA structure and function and has been engineered for biotechnology applications. Hairpin ribozymes were thought to be rare with only four known examples, which limited the interpretation of their biological importance and the starting sequences for engineering efforts. Recently, a bioinformatics approach identified hundreds of different RNA sequences in metatranscriptomic data that matched a novel permutation of the hairpin ribozyme. However, the self-cleavage activity of most of these sequences has not been experimentally demonstrated. Here, a high-throughput sequencing-based approach was used to evaluate the co-transcriptional self-cleavage activity of 855 different hairpin ribozymes in parallel. The results showed that nearly all sequences are very efficient self-cleaving ribozymes, and even rare nucleotides at highly conserved positions did not prevent observable ribozyme activity. The distribution of activity observed suggests that the metatranscriptomic sequences could contain random mutations from efficient wild-type ribozymes. The results further validate the bioinformatics approach that was used for ribozyme discovery and opens further questions about the biological roles of these ribozymes in the diverse environments where they were discovered.

biochemistry↗

bioRxiv2025

Retinal pigment epithelium-derived PD-L1 reprograms microglial cells and protects against retinal degeneration in mouse models of experimental AMD and genetic retinitis pigmentosa

Retinal degeneration is a leading cause of blindness and a significant health burden during aging worldwide. Because of its complex pathogenic mechanisms, however, effective interventions applicable to multiple retinal degenerations remain limited. Excessive microglia-mediated inflammation is a common pathological feature of different retinal degenerative diseases, and inhibiting these overactive inflammatory responses by reprogramming microglia may provide a therapeutic strategy with broad-spectrum potential. Here, we uncover a novel retinal pigment epithelium (RPE) homeostatic signal, PD-L1, that drives the reprogramming of activated microglial toward a retinal-protective phenotype. Using multiple analyses, including gene expression profiling bioinformatics, adeno-associated virus-mediated RPE-specific gene overexpression, pharmacological, and genetic manipulations, we show that RPE cells upregulate the expression of PD-L1 to modulate microglia for protection against retinal degeneration in an experimental dry AMD mouse model. Bioinformatics enrichment analysis of the high-expression gene signatures of subretinal microglia in AMD patients, combined with pharmacological blocking of NF-{kappa}B activity or using Nlrp3 knockout mice, show that PD-L1/PD-1 signaling suppresses NF-{kappa}B activation in microglia, thereby inhibiting NLRP3 inflammasome priming and attenuating microglia-mediated excessive inflammation. Furthermore, the overexpression of PD-L1 by the AAV8 virus exerts similar microglial reprogramming and retinal protective effects to alleviate retinal degeneration in a preclinical model of retinitis pigmentosa. These findings reveal that RPE-derived PD-L1 suppresses inflammatory reprogramming of subretinal microglia and mitigates retinal degeneration, highlighting a promising proof of concept for PD-L1 as a potential therapeutic target for the broad treatment of retinal degenerative diseases.

genetics↗

bioRxiv2025

Development of ENTV reverse genetics system and phenotypic evaluation of rescued virus reveals host-specific replication patterns in mosquitoes

Entebbe bat virus (ENTV) is a bat-associated flavivirus with no known vector. Research into the biology of this virus, including assessment of the possibility that it may be vector-transmitted, is hindered by a lack of molecular tools and robust genetic systems. Therefore, we sequenced the complete 3 untranslated region, which was not previously available, and developed an infectious clone of ENTV to facilitate further investigation of the virus. Virus derived from the clone replicated similarly to the parental virus isolate in various vertebrate cells. Surprisingly, ENTV replicated to high titers in Aedes aegypti and Aedes albopictus mosquito cell lines, but there was no replication or infection in Culex tarsalis cells. In addition, phylogenetic and bioinformatics analyses strongly suggested that ENTV may be associated with a mosquito host. Given the bioinformatics support and efficient growth in Aedes cells, we orally exposed Ae. aegypti and Ae. albopictus to ENTV to evaluate infection. The ENTV blood-fed mosquitoes were all negative for infection; however, when ENTV was intrathoracically inoculated, bypassing the initial midgut infection and escape barriers, it replicated to high levels in the body, without dissemination of infectious virus into the saliva. These findings suggest that, despite demonstrating high molecular compatibility at the cellular level in Aedes mosquitoes, Ae. aegypti and Ae. albopictus are unlikely to serve as competent vectors for ENTV transmission due to strong midgut infection barriers. The clone presented in this manuscript should help to clarify the mechanisms for transmission and maintenance of ENTV, which remain poorly understood.

microbiology↗

bioRxiv2025

Oxford Nanopore Sequencing as a useful tool for investigating the population dynamics of invasive begomoviruses in Sicily

Tomato yellow leaf curl disease (TYLCD) is a major viral disease severely affecting tomato crops in the Mediterranean region, leading to reduced crop yield and significant economic losses. The disease is caused by monopartite begomoviruses belonging to the Geminiviridae family, primarily tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV), which often co-infect tomato plants, promoting the emergence of recombinant viral genomes. To investigate the diversity and evolutionary dynamics of these viruses, symptomatic plants collected from agricultural sites in Sicily between 2020 and 2022, along with archived plant samples from 1994 to 1999, were analyzed. For each collection site, leaves from symptomatic plants were pooled to form representative samples. Total nucleic acids were extracted and subjected to rolling circle amplification to enrich circular viral genomes. The amplified products were sequenced using Oxford Nanopore Technologies (ONT) long-read sequencing to obtain full-length viral genomes. Bioinformatic analyses revealed that archived samples exclusively contained TYLCSV-related sequences, confirming its historical predominance in Sicilian agroecosystems. Recent samples, by contrast, no longer contained TYLCV or TYLCSV parental genomes but were dominated by TYLCV-derived recombinants such as TYLCV-IS141- and TYLCV-IS76-like variants, indicating a temporal shift in the structure of the viral population. Furthermore, a distinct group of newly emerged recombinants, provisionally referred to as TYLCV-IMS54, was identified in the most recent samples. Their genome comprises a TYLCV backbone, a 54-nucleotide segment from TYLCSV located downstream of the stem-loop region, and an 341-nucleotide region derived from TYLCV-Mild. These results demonstrate the importance of continuous viral population monitoring through ONT-based sequencing to detect emerging variants that may influence disease management strategies in tomato crops and highlight the central role of recombination in shaping begomovirus populations. IMPACT STATEMENTTomato yellow leaf curl disease (TYLCD) is one of the most damaging viral diseases affecting tomato crops in the Mediterranean basin, yet the long-term dynamics of its causal agents and the role of recombination remain challenging due to the genome plasticity of these viruses. This study provides an updated and comprehensive picture of the begomovirus population structure in Sicily, a key agricultural region for tomato production, by analyzing both contemporary and historical plant samples. Through the application of ONT long-read sequencing combined with RCA and bioinformatic analyses, this research identified persistent recombinant genotypes including a distinct group of newly emerged recombinants, named TYLCV-IMS54. These findings expand current knowledge on the genetic variability and evolutionary processes shaping begomovirus populations in Sicilian agroecosystems. The detection of recombinant genomes highlights the enduring role of recombination in begomovirus diversification. By integrating sequencing data with population and phylogenetic analysis, this work offers valuable insights into the epidemiology and management of TYLCD in regions heavily impacted or newly colonized by these viral pathogens. The study also underscores the importance of continuous molecular surveillance using ONT-based platforms to enable early detection of emerging recombinant variants, with significant implications for plant virology, crop protection and agricultural biosecurity strategies. DATA SUMMARYRaw reads are deposited in the Sequence Read Archive (SRA) of NCBI (https://www.ncbi.nlm.nih.gov/sra) with BioProjects ID PRJNA1226414 and PRJNA1273745; the recombinant TYLCV-IMS54 sequence is available in GenBank with Accession Number PQ873011. Parental TYLCV-strains used in this study could be retrieved in GenBank with the following Acc. Nos: DQ144621 for TYLCV, NC_003828 for TYLCSV, KJ913682 for TYLCV-Mild. Recombinant strains could be retrieved in GenBank with the following Acc. Nos.: LN846609 for TYLCV-IS76 and AF271234 for TYLCMaV. The authors confirm that all supporting data, code and protocols have been provided within the article or through supplementary data files.

plant biology↗

bioRxiv2025

Thermophilic bacteria employ a contractile injection system in hot spring microbial mats

Bacterial contractile injection systems (CISs) are multiprotein complexes that facilitate the bacterial response to environmental factors or interactions with other organisms. Multiple novel CISs have been characterised in laboratory bacterial cultures recently; however, studying CISs in the context of the native microbial community remains challenging. Here, we present an approach to characterise a new, bioinformatically predicted CIS by directly analysing bacterial cells from their natural environment. Using cryo-focused ion beam milling and cryo-electron tomography (cryoET) imaging, guided by 16S amplicon sequencing, we discovered that thermophilic Chloroflexota bacteria produce intracellular CIS particles in a natural microbial hot spring mat. We then found a niche-specific production of CIS in the structured microbial community using an approach combining shotgun metagenomics, proteomics, and immunogold staining. Bioinformatic analysis and imaging revealed CISs in other extremophilic Chloroflexota and Deinococcota. This Chloroflexota/Deinococcota CIS lineage shows phylogenetic and structural similarity to previously described cytoplasmic CIS from Streptomyces and probably shares the same cytoplasmic mode of action. Our integrated environmental cryoET approach is suitable for discovering and characterising novel macromolecular complexes in environmental samples.

microbiology↗

bioRxiv2025

Integrated Expression Analysis of C-MYC Oncogene-Associated Pathways in Gastric Adenocarcinoma and its Correlation with Clinicopathological Factors

BackgroundThe C-MYC oncogene is a well-established driver of gastric carcinogenesis, yet the integrated expression pattern of its complex regulatory network and its clinical implications in gastric adenocarcinoma (GAC) remain to be fully elucidated. This study aimed to perform an integrated bioinformatic analysis of C-MYC and its associated pathways in a cohort of GAC patients to delineate its expression profile, assess its potential as a biomarker, and correlate its patterns with clinicopathological factors such as Lauren classification and neoadjuvant treatment status. MethodsThe study included transcriptome data from 74 GAC tumor samples and 68 normal gastric tissue samples. Following RNA sequencing, a comprehensive bioinformatic analysis was conducted on a curated list of 21 C-MYC-associated genes. The methodology included differential expression analysis (DESeq2), unsupervised hierarchical clustering, Principal Component Analysis (PCA), and Receiver Operating Characteristic (ROC) curve analysis to evaluate diagnostic performance. Gene expression levels were also statistically correlated with Lauren histological subtypes and neoadjuvant therapy status using the Wilcoxon rank-sum test. ResultsThe analysis revealed a profound dysregulation of the C-MYC network in GAC. While MYC itself was significantly upregulated, its transcriptional antagonists, particularly MXD4 and MXD3, were the most significantly downregulated genes. This gene signature robustly separated tumor from normal tissues in both hierarchical clustering and PCA. ROC analysis demonstrated the outstanding diagnostic potential of several genes, with MXD4 achieving a perfect Area Under the Curve (AUC) of 1.00, surpassing the diagnostic value of MYC (AUC=0.86). Stratification by Lauren classification showed that MYC and its stability regulator PTBP1 were significantly more expressed in the intestinal subtype, whereas the repressors MXD3 and MXI1 were higher in the diffuse subtype. No significant expression differences were observed based on neoadjuvant treatment status. ConclusionGastric adenocarcinoma is characterized by a coordinated dysregulation of the C-MYC network, marked by both oncogene activation and a concurrent loss of its key transcriptional repressors. The profound downregulation of antagonists like MXD4 serves as an exceptionally accurate molecular signature for GAC, suggesting its potential as a diagnostic biomarker superior to MYC alone. The divergent expression patterns between Lauren subtypes highlight distinct molecular pathobiology and may have implications for targeted therapies

genetics↗

bioRxiv2025

Nuclear genome profiling of two Mexican orchids of the genus Epidendrum

Characterizing genomic properties such as genome size, ploidy level, heterozygosity, and repetitive DNA proportion and composition without relying on genome assembly is crucial for profiling the genomes of non-model species. Little is known about the nuclear genome of the large neotropical orchid genus Epidendrum. This study compares genome profiles of Epidendrum anisatum and Epidendrum marmoratum, using flow cytometry and k-mer analysis approaches, as well as bioinformatics ploidy level estimation and repeatome characterization. Multiple depths of coverage, k values, and k-mer-based tools for genome size estimation were explored and contrasted with cytometry genome size estimations. Cytometry and k-mer analyses yielded a consistently higher genome size for E. anisatum (mean 1C genome size = 2.59 Gb) than E. marmoratum (mean 1C genome size = 1.13 Gb), which represents a 2.3-fold genome size difference. Both species were identified as diploid with no evidence of strict partial endoreplication. The most important aspects to be taken into account to improve genome size estimation were heterozygosity, depth of coverage, and the maximum k-mer coverage. The genomes of both species were found to be highly repetitive (63-73%) and heavily dominated by Ty3-gypsy retrotransposons, particularly those of the Ogre family. Additionally, the genome of E. anisatum was characterized by the presence of a 172 bp satellite (AniS1), which represented 11% of the genome size. Together, both Ty3-gypsy transposons and AniS1 shape the genome size difference between the two genomes. This study provides the first genome profiling for species in the genus Epidendrum, but also highlights the importance of using flow cytometry, cytogenetic approaches and bioinformatics techniques in combination for genome profiling.

genomics↗

bioRxiv2025

Non-Invasive Diagnostic Evaluation of Urinary Exosomal Let-7c Cluster Expression in Bladder Cancer Using Machine Learning Approaches

BackgroundBladder cancer (BCa) diagnosis typically relies on invasive cystoscopy, which is effective but costly and uncomfortable. Urinary microRNAs (miRNAs), especially exosomal ones, are promising non-invasive biomarkers due to their stability in biological fluids and disease specificity. However, challenges such as population variability, methodological inconsistencies and normalization issues hinder their clinical translation, emphasizing the need for innovative approaches to enhance diagnostic performance. ObjectiveTo evaluate the diagnostic potential of urinary exosomal let-7c cluster (let-7c-5p, miR-99a-5p and miR-125b-5p) in BCa patients by integrating miRNA expression data with Machine Learning (ML) models. MethodsUrine samples were collected from 66 participants, including 50 BCa patients and 16 healthy controls (HC). Exosomal miRNAs were isolated and quantified using Quantitative Real-Time-Polymerase-Chain-Reaction (qRT-PCR). Statistical analysis and hypothesis tests were conducted to explore the nature and diagnostic relevance of individual biomarkers. A logistic regression classifier was applied to evaluate both the combined and differential diagnostic capabilities of the selected biomarkers. Accuracy, precision, recall and AU-ROC scores were used to assess model performance. Bioinformatics analysis was performed to identify pathways associated with the features prioritized by the ML models, ensuring their relevance to BCa. ResultsThe result revealed significant differentiation between BCa patients and HC, with miR-99a-5p (p=0.013, AU-ROC=0.71) and miR-125b-5p (p=0.047, AU-ROC=0.64) demonstrating reliable diagnostic performance (let-7c-5p showed weaker discrimination, AU-ROC=0.65, p>0.1). The logistic regression ML model achieved an accuracy of 80.0% (AU-ROC=0.86, recall=100%) in distinguishing cancer from HC and 53.3% (AU-ROC=0.63) when applied to miRNA-only grade classification. When clinical variables were integrated with miRNA expression, performance improved to 73.3% accuracy (AU-ROC=0.61) for high-versus low-grade differentiation. Across Ta-T2, miR-99a-5p displayed relatively better separation, whereas let-7c-5p and miR-125b-5p showed weak stage-related differences. The integration of bioinformatics analysis confirmed the biological relevance of these miRNAs in BCa-related pathways, including PI3K-Akt, p53, NF-{kappa}B and RAS/MAPK signaling, with hub genes such as TP53, MYC, EGFR, and CCND1 identified, further validating the diagnostic utility of the selected biomarkers. ConclusionUrinary let-7c cluster miRNAs demonstrate promising diagnostic potential when analyzed with ML models, offering a non-invasive alternative to conventional methods. These findings highlight the promise of ML-based approaches alongside molecular markers for advancing clinical diagnostics in BCa.

molecular biology↗