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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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300 records · Page 17Linked to original sources

bioRxiv2026

Kaposi's sarcoma-associated herpesvirus forms and maintains R-loops at origins of lytic replication

GC-rich sequences are abundant in human herpesviruses genomes. GC-rich regions can form three-stranded RNA:DNA hybrid structures called R-loops. Though these hybrid structures serve important biological roles at telomeres or during cellular DNA synthesis, unscheduled or prolonged R-loop formation causes DNA damage and genome instability. For this reason, several mechanisms exist to resolve R-loops including endoribonucleases RNaseH1 (constitutively expressed) and RNaseH2A (cell cycle-regulated) which degrade the RNA portion of the R-loop. The Kaposi's sarcoma-associated herpesvirus (KSHV) origins of lytic replication (OriLyts) contain multiple cis-acting elements that are required for viral DNA replication including the production of GC-rich and repetitive transcripts, T1.4 (OriLyt-L) and kaposin (OriLyt-R). We previously showed that R-loops form at both OriLyts and that deleting kaposin repeats or decreasing their GC-rich content prevented R-loop formation at OriLyt-R, reduced genome amplification after primary infection and caused defects in latency establishment. To define the contribution that R-loops play in KSHV replication, we overexpressed RNaseH1, reasoning that excess RNaseH1 would resolve both OriLyt R-loops. However, RNaseH1 protein levels decreased following KSHV reactivation in both iSLK and BCBL-1 cell lines. Using co-transfection, we discovered that the KSHV viral replication and transcription activator protein, RTA, mediated RNaseH1 protein decreases in a E3 ligase domain-dependent manner without impacting levels of its cognate RNA transcript. We attempted to construct an RTA-resistant yet functional version of RNaseH1 by site-directed mutagenesis of lysine residues individually or in combination, yet these constructs remain susceptible to RTA-mediated protein decreases. An amino terminally tagged RNaseH1 displayed reduced susceptibility to RTA, suggesting that RTA may target the N-terminus of RNaseH1 for ubiquitination. However, overexpression of the cell-cycle regulated endonuclease, RNaseH2, exhibited RTA resistance, suggesting RNaseH2 may be a tool that will effectively resolve R-loops during KSHV infection. KSHV is not the only herpesvirus to encode a protein that reduces RNaseH1 levels, as co-expression of RTA homologs from the related gamma-herpesviruses EBV and MHV-68 likewise decreased steady-state levels of RNaseH1 protein. We propose that RTA-mediated RNaseH1 degradation is conserved strategy to ensure R-loop persistence during gamma-herpesvirus infection, underscoring the importance of these structures.

microbiology↗

bioRxiv2026

Designing antimicrobials with programmable mechanism and safety

Antimicrobial peptides (AMPs) are a promising solution to antimicrobial resistance, yet generative models for their design cannot control the physicochemical properties and motifs that shape activity and selectivity. Here, we present OmegAMP, a conditional diffusion framework controlling net charge, mean hydrophobicity, and sequence length, supporting de novo, analog, and motif-guided design. Across 204 wet-lab characterized peptides, de novo generation yielded antimicrobials with broad activity against multidrug-resistant Gram-negative isolates. Analog generation converted six inactive prototypes into antimicrobials, with the prototype determining each analog's membrane-disruption mode and mammalian-cell safety. Motif-guided analog generation preserved lipopolysaccharide engagement of active prototypes, and a redesigned non-antimicrobial leucine zipper acquired antimicrobial activity while retaining DNA-perturbing character in vitro. In murine skin and thigh infection models, leads reduced bacterial burden, with a motif-guided DNA-perturbing lead matching the fluoroquinolone control systemically. OmegAMP opens a programmable route to new peptide antibiotics whose mechanism and safety follow from the chosen prototype.

bioinformatics↗

bioRxiv2026

Dog-wise canine gut metagenome assemblies with reconstructed bacterial genomes and viral candidates

Long-read metagenomic sequencing can improve genome recovery from complex gut microbial communities, yet directly reusable canine gut genome resources remain limited. Here we describe DogMAG, a canine gut metagenome resource based on dog-wise long-read and hybrid assemblies generated by grouping sequencing libraries according to canonical dog identity before assembly. The final dataset comprises 41 assemblies linked to 277 FASTQ records, including 30 Flye long-read-only and 11 OPERA-MS hybrid assemblies. A single integrated BASALT workflow produced 11,276 selected bin/version records, followed by explicit quality-based re-selection of 3,418 medium-quality-or-better metagenome-assembled genome candidates. External dRep dereplication yielded 792 strain-like representatives at 99% average nucleotide identity and 135 species/SGB-like representatives at 95%. GTDB-Tk classified all 792 representatives as Bacteria. Viral screening identified 22,068 geNomad predictions, of which 3,374 Complete, High-quality or Medium-quality viral/proviral candidate rows passed CheckV filtering with contamination [≤]10%. DogMAG provides assemblies, genome and viral candidate sequences, metadata, provenance tables and workflow scripts for reuse, benchmarking and reanalysis.

microbiology↗

bioRxiv2026

Taxonomic classification cost tracks neither sequencing depth nor community richness at single-sample scale: a measured resource protocol for 16S rRNA amplicon pipelines

Marker-gene amplicon workflows are routinely run on shared compute, yet the cores, memory and wall time they are given are chosen by convention and not by measurement. We present a protocol for measuring them, applied to the two dominant stages of a QIIME 2 16S rRNA pipeline, DADA2 denoising and Naive Bayes taxonomic classification, across nine upper-respiratory samples from a paediatric otitis media cohort. The two stages do not consume the same input: denoising reads every sequence, classification only those surviving it. Subsampling one library across a 27-fold range of sequencing depth, denoising wall time rose 14.3-fold while classification changed by 1% and its peak memory not at all (3.11 GiB). Amplicon sequence variant (ASV) richness rose 2.8-fold over that range, so this is not richness saturating: the stage is dominated by a fixed per-invocation cost. Across a body-site gradient of 5 to 70 ASVs, denoising followed read count (exponent 0.75) while classification followed neither: a 5-ASV effusion and a 70-ASV adenoid community cost 40.81 s and 40.79 s. One ASV took 36.20 s and 218 took 37.27 s, 97% fixed cost. Thread-level parallelism offered little benefit. Denoising peaked at 1.18x near 8 threads and then declined; classification was slower at every setting above one job, consuming 10.5 times the CPU at 40. Representative sequences and their taxonomic assignments were identical at 1, 4 and 40 threads, so a reduced allocation changes what the analysis costs, not what it reports. Extending the query set to 10,000 sequences located two distinct boundaries: eight jobs first beat one at roughly 5,000 queries, and fitted fixed and per-query costs become equal at 15,248. Both lie roughly two orders of magnitude above the richest single sample measured. Practically: size denoising by read count, calibrate classification once against the reference in use, request one job for classification below a few thousand sequences, and take throughput from sample-level parallelism. Protocol, data and analysis code are released with the pipeline.

bioinformatics↗

bioRxiv2026

Dehydration triggers anomalous subdiffusion in biomimetic cell membranes

Lipid diffusion plays a central role in shaping the structural organization of cell membranes, maintaining lipid homeostasis, and facilitating cellular transport and signaling. The lateral mobility of phospholipids in membranes depends heavily on their hydration state. Furthermore, the activation energy of diffusion increases in conditions of reduced membrane hydration, suggesting that the underlying diffusion mechanism changes upon dehydration. Using two variants of fluorescence correlation spectroscopy (point FCS and scanning FCS) and two membrane reporters, we demonstrate that mild dehydration of phase-separated biomimetic cell membranes alters the lipid diffusion mechanism, resulting in anomalous subdiffusion rather than free Brownian motion. Importantly, the anomalous diffusion parameter, , decreases significantly upon the initial reduction of the membrane hydration layer, and the effect is fully reversible upon rehydration. These observations strongly indicate the reversible shift in lipid diffusion mode rather than irreversible membrane damage. We propose that this anomalous subdiffusion is caused by the formation of temporarily immobile lipid pockets in the membrane upon dehydration. These results therefore provide important insights into the mechanism of lipid diffusion in membranes undergoing local and transient dehydration, which is an important intermediate step in various biological processes associated with membrane fusion, such as neurotransmission, fertilization, and viral entry.

biophysics↗

bioRxiv2026

Full-length 16S profiling reveals individualized gut microbiota dynamics during short-duration spaceflight

Human spaceflight may perturb the gut microbiota, but densely sampled short missions remain poorly characterized. We profiled 27 phase-matched fecal samples from two astronauts during an 18-day International Space Station mission and one ground-based participant following the same daily schedule using Oxford Nanopore full-length 16S sequencing. Participant identity dominated genus-level Bray-Curtis variation (R2 = 0.489, p < 0.001). In astronaut-only community analyses, mission phase explained 23.4% of genus-level (p = 0.035) and 22.3% of species-level (p = 0.021) variation. Astronauts showed greater displacement from personal baselines than B1 (0.331 versus 0.171) and 1.58-fold higher volatility. Astronaut-only taxon models identified 2 of 81 genera and 5 of 139 species; Collinsella increased from quarantine to orbit (coefficient = 2.586, q = 0.037). Thus, the short-duration spaceflight interval was accompanied by individualized, temporally localized community and taxon shifts rather than uniform microbiota restructuring.

microbiology↗

bioRxiv2026

High-Throughput, automated assay for detection of colonization by Candida auris

Candida auris is an emerging multidrug-resistant fungal pathogen associated with healthcare-associated outbreaks, persistent colonization, and invasive infections. Increasing demand for surveillance has created a need for high-throughput methods capable of supporting large-scale screening programs. We developed and validated an automated laboratory-developed real-time PCR assay for detection of C. auris colonization on the Hologic Panther Fusion(R) open-access platform and compared its performance with the existing BD MAX assay. Analytical performance was evaluated by assessing limit of detection, accuracy, precision, specificity, inclusivity, reproducibility, and reagent and specimen stability. The Panther Fusion(R) assay demonstrated a limit of detection of approximately 18 CFU/reaction and showed 97% overall agreement with the BD MAX assay. Positive and negative percent agreement were 94% and 100%, respectively, with excellent agreement between methods ({kappa} = 0.94). No cross-reactivity was observed with non-C. auris organisms, all five major C. auris clades were detected, and assay performance remained stable across operators, reagent and specimen storage conditions. Following implementation, 26,838 clinical specimens were tested on the Panther Fusion(R) platform. Retrospective analysis demonstrated lower equivocal (0.28%) and indeterminate (0.09%) rates than those observed on the ABI and BD MAX platforms. Among PCR-positive specimens that underwent culture, the Panther Fusion(R) assay demonstrated 87.24% culture agreement. Because retrospective data were collected during different testing periods and patient populations, comparisons between platforms were not used to assess relative assay sensitivity or specificity. Implementation of the Panther Fusion(R) assay increased surveillance testing capacity from approximately 88 to 500 specimens per shift while maintaining robust analytical performance.

microbiology↗

bioRxiv2026

Basophilic Erythroblast Emerges as the Key Turning Point in Polycythemia Vera

Abstract Polycythemia vera (PV) is a rare, chronic myeloproliferative neoplasm driven by the JAK2V617F mutation and characterized by uncontrolled erythroid proliferation. Although the mutation arises in hematopoietic stem cells, the differentiation stage at which its transcriptional consequences first become biologically meaningful has remained undefined. Using a multi-layer transcriptomics integration approach that combined differential gene expression, NicheNet ligand-receptor analysis, pseudotime trajectory inference, and CNV profiling on scRNA seq data, alongside bulk transcriptome validation, we identified basophilic erythroblasts as the critical transition point at which JAK2V617F shifts from a genomically present but transcriptionally silent state to an actively trajectory-altering and treatment-responsive disease driver. Differential expression revealed a qualitatively distinct disease signature at this stage, including ERFE-mediated iron dysregulation, MAP2K2-driven RAS/MAPK co-activation, and epigenetic reprogramming. NicheNet showed the establishment of a TGF{beta} superfamily and chemokine-driven niche-remodeling axis, and pseudotime analysis demonstrated that basophilic erythroblasts are the first erythroid population to exhibit condition-dependent trajectory divergence, whereas earlier progenitors showed none despite carrying the mutation. Interferon- treatment showed its broadest counterresponse at this stage but declined sharply thereafter, identifying basophilic erythroblasts as both the principal therapeutic target and the point of maximum vulnerability in PV.

bioinformatics↗

bioRxiv2026

Structures of LolB bound to LolA or lipoprotein resolve the final steps of bacterial lipoprotein trafficking

In Gram-negative bacteria, lipoproteins are structural elements of the outer membrane and essential components of machineries responsible for its construction and maintenance. The Lol system, responsible for the trafficking of lipoproteins from the site of maturation on the inner membrane to the outer membrane, is therefore crucial to the function of the cell envelope and a key target of efforts to find novel antimicrobials. In the final steps of this process, the outer membrane receptor, LolB accepts triacylated lipoproteins from the periplasmic chaperone LolA before inserting them into the outer membrane. Here we present a structure of LolB in complex with LolA, validated by in vivo and in vitro assays, highlighting how positively charged residues on the convex face of the LolB {beta}-barrel underpin complex formation. A protruding loop of LolB, essential for function, inserts into the LolA cavity in position to initiate the displacement of substrate lipoprotein from LolA to enable transfer to LolB. Structural resolution of a lipoprotein-bound LolB complex in combination with biophysical assays shows how a molecular latch releases the lid of the cavity to accommodate the lipoprotein acyl chains. Modelling of these structures onto computationally predicted orientations for LolB on the outer membrane provides a rationale for LolA release and lipoprotein triacyl group membrane insertion. Taken altogether, our data elucidate atomic resolution of two key intermediates and provide a greater understanding of the terminal steps of lipoprotein trafficking events at the bacterial outer membrane.

microbiology↗

bioRxiv2026

Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli

Background Targeted-Antibacterial Plasmids (TAPs) are engineered mobile genetic elements that use bacterial conjugation to deliver selective CRISPR/Cas9 antibacterial activity against a specific target strain. Yet, the efficiency of TAPs is typically evaluated at a single time point, whereas the success of TAP-mediated resensitization critically depends on the dynamics of plasmid transfer and the complex interactions between bacterial subpopulations. This is the first study to evaluate the efficiency of a conjugation-based antibacterial approach at the subpopulation level, using an analytical framework analogous to that used for conventional antibiotics. Here, we investigate which process limits resensitization by TAPF-dCas9-OXA48: plasmid delivery, dCas9 activity, or the emergence of refractory and escape populations. Methods We fitted a mechanistic model of five interacting subpopulations (donors, recipients, transconjugants, escapers, and recusants) to 44 longitudinal conjugation experiments and used the fitted model to explore a range of biologically relevant scenarios. Results Using longitudinal conjugation data spanning 24 h, we show that up to 24% of recipients become recusants within 24h, refractory to further conjugation via entry exclusion, while secondary transconjugant emergence stays below 0.01%. Overall resensitization efficiency reaches up to 80%. Conclusion Plasmid transfer, rather than dCas9 repression, therefore appears to be the main bottleneck limiting the efficiency of TAPF-dCas9-OXA48 efficiency. These results identify plasmid delivery as a key engineering target for improving the performance of future TAPs.

bioinformatics↗

bioRxiv2026

Immunogen selection and prior immunity shape antibody breadth following immunisation with avian H5 hemagglutinin

Avian influenza A viruses pose a persistent zoonotic threat to humans owing to their expanding host range and high case fatality rates. In particular, viruses from the 2.3.4.4b clade of the H5 subtype have now been detected in over 60 mammalian species, raising serious pandemic concerns. Understanding immune recognition of the H5 hemagglutinin (HA) is therefore critical for effective vaccine design and pandemic preparedness. To understand the breadth of cross-recognition induced by different H5 strains, we selected genetically diverse H5 human isolates from 2003-2023 and assessed neutralising antibody responses elicited by adjuvanted recombinant HA protein-based vaccines in C57BL/6 mice. Neutralisation activity of sera was determined against seven H5 HA variants using pseudotyped viruses and a PR8-reassortant virus in micro-neutralisation assays. Our results showed a wide variety of cross-strain neutralisation across H5 HA antigen variants. The conventional vaccine strain A/Indonesia/05/2005 displayed narrow activity against emerging clade 2.3.4.4b viruses, whereas ancestral variants exhibited cross-neutralisation profiles showing a diversity of breath but with limited potency. Polyvalent H5 HA formulations and nanoparticle-displayed H5 HA platforms substantially broadened cross-neutralisation against diverse H5 strains. To examine the impact of pre-existing immunity on H5 vaccine immunogenicity in mouse models, mice were primed with either seasonal influenza infection or quadrivalent influenza vaccine (QIV) prior to H5 HA immunisation. QIV pre-vaccination, but not prior influenza infection, enhanced subsequent neutralizing responses towards A/Fujian-Sanyuan/21099/2017 (clade 2.3.4.4b) H5. Collectively, our results demonstrate that immunogen selection and prior immunity shape antibody breadth following immunisation with avian A(H5) hemagglutinin.

immunology↗

bioRxiv2026

Exploratory multi-omics analysis reveals sex-specific differences in microbial response to antibiotic exposure

Antibiotic exposure is a major driver of microbiome disruption and antimicrobial resistance gene (ARG) expansion. Yet, the role of biological sex in shaping these responses remains poorly understood. Most studies do not stratify antibiotic-induced microbiome changes by sex or integrate multi-omics datasets, limiting our understanding of how microbial, metabolic, and immune responses interact. Therefore, there remains a critical need for an integrative systems-level approach to determine how sex-specific disruptions under antibiotic pressure are paralleled across microbial, metabolic, and host immune layers. The objective of this work was to perform an exploratory study investigating how continuous antibiotic exposure reshaped the gut microbiome across sexual maturation and how these perturbations influenced downstream host responses in a sex-specific manner using an integrative multi-omics framework. Male and female mice that were exposed to continuous antibiotics were profiled over sexual maturation using shotgun metagenomics, untargeted metabolomics, and bulk RNA sequencing of the spleen to assess microbial composition, ARG dynamics, metabolic profiles, and immune responses. Overall, our results demonstrated sex-specific correlations at a systems-level that help provide valuable context to the differences observed in males and females upon antibiotic exposure.

microbiology↗