bioRxiv Subject Collection: All

This feed contains articles for bioRxiv Subject Collection "All"

APOBEC3 antagonism fully explains HIV-1 Vif essentiality under interferon and differentiation conditions

HIV-1 virion infectivity factor (Vif) counteracts APOBEC3 (A3) proteins by targeting them for proteasomal degradation, thereby preventing lethal G-to-A mutations and loss of infectivity. However, Vif also degrades additional cellular proteins, raising the possibility that its essential role in infectious virion production may extend beyond A3 antagonism, particularly under inflammatory or differentiation conditions. Whether such conditions reveal additional essential Vif targets remains unresolved. Here, using interferon (IFN)-stimulated or phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytic cells, we directly addressed this question. Although type I IFN and PMA markedly suppressed viral production through Vif-independent mechanisms, {Delta}Vif viruses produced from stimulated parental cells exhibited severely reduced infectivity. In contrast, disruption of A3A-A3G fully restored {Delta}Vif infectivity to wild-type levels under all conditions tested. G-to-A mutations were attributable exclusively to A3 proteins under both IFN and PMA stimulation, whereas IFN-induced, A3-independent blocks to reverse transcription were not antagonized by Vif. Across diverse HIV-1 strains, the requirement for Vif was strictly dependent on A3 family proteins. These findings demonstrate that the essential role of Vif is fully explained by antagonism of A3-mediated restriction and that evolutionary pressure on Vif is driven predominantly by the need to counteract A3-mediated restriction and mutagenesis.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.719124v1?rss=1

Shimizu, R., Jonathan, M., Terasawa, H., Saito, A., Monde, K., Ikeda, T.

Convergent antigenic drift of the influenza hemagglutinin lateral patch across time and species

The lateral patch epitope of the H1 hemagglutinin (HA) was a dominant target of antibodies following exposure to the 2009 pandemic H1N1 virus. However, the conservation and potential for antigenic drift in the lateral patch remain unresolved. Here, we used lateral patch-specific monoclonal antibodies (mAbs) to understand the antigenicity of the lateral patch of human, avian, and swine H1Nx viruses spanning from 1918 to 2022. We identified discrete mutations that evaded lateral patch-targeting mAbs in pre- and post-2009 H1N1 viruses, leading to genetic differences in lateral patch-targeting antibodies in individuals across birth years. We observed that the lateral patch remains well conserved across zoonotic sources, suggesting existing lateral patch antibodies could protect against a future H1Nx pandemic. Together, these data support that lateral patch antigenic drift has shaped the human B cell repertoire against influenza viruses and that the lateral patch remains an attractive target for pandemic preparedness.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718966v1?rss=1

Lemus-Reyes, J. I., Fernandez-Quintero, M. L., Ayala, E., Swanson, O., Good, M., Ji, W., Suja, D., Han, J., Ward, A., Guthmiller, J.

155 years after Van Beneden: redescription and first molecular characterisation of the enigmatic type species, Ascarophis morrhuae Van Beneden, 1870 (Nematoda, Cystidicolidae), and comparison to other Ascarophis species in the North Atlantic

Nematodes belonging to the Cystidicolidae Skrjabin, 1946 constitute more than 23 genera of 111 recognized species in fish from many habitats including the deep-sea, continental shelves, estuarine and freshwater habitats. The taxonomy of many species within the Cystidicolidae is unsettled due to their small size and correspondingly small morphological characters requiring use of scanning electron microscopy and supported more recently by molecular studies. The type species, Ascarophis morrhuae Van Beneden, 1870, which belongs to one of the first described and most speciose cystidicolid genera with 46 species, is based on a two-sentence description of a single female specimen from an Atlantic cod, Gadus morhua, presumably captured off the coast of Belgium in the North Sea (Van Beneden, 1870). New material was collected/examined from Atlantic cod and haddock, Melanogrammus aeglefinus, from Iceland and the North Sea and specimens present in the Natural History Museum, London were also studied. Based on these materials, A. morrhuae is morphologically redescribed and the first DNA sequences of this species are provided, it is differentiated from other Ascarophis species present in the North Atlantic and previous records are reviewed. This information provides a foundation for taxonomic and phylogenetic reconsideration of all cystidicolid nematodes and related families.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718624v1?rss=1

Appy, R. G., Vanhove, M. P. M., MacKenzie, K., Hernandez-Orts, J. S., Kmentova, N.

GANGE: Achieving Sequencing Without Sequencing With Diffusion Guided Generative Genomic Transformer

The genome of a species is its book of life, but opening that book remains a costly affair due to the limitations the existing sequencing technologies pose. Short reads sequencers struggle to capture long and complex genomes, though have high fidelity rate. To counter that long reads from IIIrd generation sequencers are used, which are full of indel errors. Thus, reads from both approaches are collectively used with very high coverage, making the sequencing projects unreasonably high of cost and unapproachable to majority. Here we present a first of its kind generative deep-learning system, GANGE, which not just recovers the correct sequence with high accuracy from indel prone ONT reads at manifold lesser coverage but also extends it by 4kb, achieving sequencing without sequencing, horizontally as well as vertically while maintaining >92% accuracy consistently. This all makes it possible to drastically pull down sequencing project cost. GANGE was tested across A. thaliana, O. sativa genomes and Human chromosome 1 where it delivered outstanding assembly performance. Besides this, it was also used to accurately generate 2kb upstream promoters of all the genes from 12 different species, demonstrating that one can now also take up regulomics research just using RNA data alone when genome sequence is not available. With this all, GANGE brings a democratic turning point in the area of genomics and sequencing research.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718133v1?rss=1

Gupta, S., Kumar, A., Bhati, U., Shankar, R.

cellNexus: Quality control, annotation, aggregation and analytical layers for the Human Cell Atlas data

Large-scale single-cell atlases such as the Human Cell Atlas have transformed our understanding of human biology. Yet, the lack of a robust framework that standardises quality control, expands cellular annotation, and adds normalisation and analytical layers, limits multi-study analyses and the usefulness of this resource. Here we present cellNexus, a comprehensive tool and resource that converts the Human Cell Atlas collection into analysis-ready data by linking quality control layers, metadata enrichment, expression normalisation, analysis and data aggregation. These enhancements enable robust statistical modelling across studies, exemplified by a multi-tissue map of immune cell communication during ageing, which reveals macrophage-muscle axes as among the most depleted regenerative interactions with age. All harmonised layers, including pseudobulk and cell-cell communication summaries, are accessible via a public web interface and with R and Python APIs. By providing continuous integration with CELLxGENE releases, cellNexus transforms large cell atlas corpora into an accessible, reproducible, interoperable foundation for large-scale biological discovery and the next generation of single-cell foundation models.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.14.718336v1?rss=1

Shen, M., Gao, Y., Liu, N., Bhuva, D., Milton, M., Henao, J., Andrews, J., Yang, E., Zhan, C., Liu, N., Si, S., Hutchison, W. J., Shakeel, M. H., Morgan, M., Papenfuss, A. T., Iskander, J., Polo, J. M., Mangiola, S.

Bacteria-mimetic bioadhesives with multivalent mucoadhesion and drug-compatible delivery

The ability to adhere to mucus-lined tissues underpins a range of biomedical devices and therapies. However, many existing strategies rely on covalent bonding chemistries and can be unstable, cytotoxic, or incompatible with therapeutics. Here, we present a bacteria-mimetic bioadhesion strategy inspired by Vibrio cholerae. A short Bap1-derived adhesion peptide is grafted onto chitosan to strengthen mucus interactions through multivalent, cooperative secondary bonding, while preserving pH-triggered interfacial bridging behavior. Bacterial peptide grafting significantly increases adhesion energy on porcine intestine, and when paired with a tough hydrogel matrix achieves adhesion energies >400 J/m^2 without forming covalent bonds to tissue. Confocal imaging reveals deep tissue penetration (~80 m) with markedly enhanced mucin binding and no loss of cytocompatibility. Ex vivo intestinal delivery and in vitro drug release tests demonstrate improved drug transport and tissue exposure compared to carbodiimide-mediated covalent bonding strategy. These findings establish a bacteria-mimetic bioadhesion strategy for tissue repair and drug delivery. Novelty Statement: Bioadhesive designs have drawn inspiration from nature such as mussel-inspired catechol chemistry and gecko-inspired dry adhesion. In contrast, bacterial adhesion mechanisms, despite enabling robust colonization of mucus-lined tissues under demanding conditions, remain largely overlooked. This work introduces a bacteria-mimetic bioadhesion strategy that selectively repurposes a short, non-pathogenic peptide derived from a Vibrio cholerae adhesin to enhance bioadhesion through multivalent, cooperative physical interactions rather than covalent bonding. This strategy significantly toughens adhesion even on chitosan, a polymer already rich in adhesive functional groups. By decoupling bacterial adhesion function from pathogenic risk, this study establishes bacterial adhesion peptides as a safe, modular, and mechanistically distinct foundation for next generation bioadhesives with improved drug compatibility.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718429v1?rss=1

Hohnston, E., Farrow, M., Yang, Z., Bahmani, A., Liu, Y., Huang, X., Yan, J., Li, J.

ARMH3 acts as a central scaffold at the Golgi/TGN through interactions with Arl5, GBF1, and PI4KB

The armadillo repeat protein ARMH3 regulates the activity and localisation of the Golgi resident lipid kinase phosphatidylinositol 4 kinase III{beta} (PI4KB) and the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) that activates Arf1. ARMH3 localises to the trans Golgi network (TGN) via the GTPase Arl5. We used hydrogen deuterium exchange mass spectrometry (HDX-MS) and AI-enabled modeling to define the interfaces of ARMH3 with its binding partners Arl5, PI4KB, and GBF1. The ARMH3-Arl5 interface was determined to consist of the N and C termini of ARMH3, with Arl5 binding causing conformational changes in ARMH3 located at a shared PI4KB/GBF1 interface. Both PI4KB and GBF1 form mutually exclusive complexes with ARMH3, with GBF1 binding to ARMH3 through a disordered loop we have named the ARMH3 binding region (ABR). The ARMH3 interfaces in PI4KB and GBF1 contain phosphosites, with the phosphomimetic mutation of GBF1 blocking complex formation. These findings provide new insights into the role of ARMH3 as a master coordinator of GTPase and phosphoinositide signaling at the Golgi/TGN.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718671v1?rss=1

Scott, M. K., Klynsoon, G. C., Walsh, E. E., Suresh, S., Nyvall, H. G., Burke, J. E.

Dorsal fins are not universal stabilizers in cetaceans: limited yaw effects and flipper-coupled roll stability

Cetacean dorsal fins are traditionally regarded as vertical stabilizers for yaw and roll, yet marked variation in fin area and position suggests that this function is not universal. We combined computational fluid dynamics simulations of five cetacean species with comparative analyses of dorsal fin and flipper morphology across 81 species to test whether variation in dorsal fin morphology reflects the evolution of hydrodynamic stabilization. Yaw-destabilizing moments were dominated by the trunk regardless of flipper posture, whereas dorsal fins were generally too small and too close to the center of rotation to provide substantial static yaw restoration. In contrast, dorsal fins influenced roll stability in concert with extended anhedral flippers. Although dorsal fins were likely present in the common ancestor of extant cetaceans, strong dorsal fin-mediated roll stabilization was largely restricted to oceanic bite-feeding delphinids, in which rapid evolutionary enlargement of the dorsal fin and persistently extended anhedral flippers likely enhanced roll stability. In most other lineages, roll stability could be maintained by flipper posture alone despite small dorsal fins. These results recast the cetacean dorsal fin not as a universal stabilizer, but as a lineage-specific roll stabilizing structure whose function emerges through mechanical coupling with the flippers.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718588v1?rss=1

Okamura, T., Maeda, M., Nishimura, F., Yoda, K.

PREFERENTIAL INVASION OF DIFFERENTIATED BLADDER CARCINOMA CELLS BY FLAGELLATED GROUP B2 ESCHERICHIA COLI

Phylogenetic group B2 Escherichia coli is associated with urinary tract infections and other pathologies, but the basis for this phylogenetic skew is not understood. One aspect of urinary tract infections is binding to and entering uroepithelial cells. To test whether a phylogenetic skew exists for cell invasion, we examined invasion of 10 E. coli strains from three phylogenetic groups into CRL2169 and HTB-9 cells, which are derived from grade 1 and grade 2 bladder carcinomas, respectively. The top four strains that invaded CRL2169 were from group B2: three of these strains had more flagella gene transcripts than the other seven strains. The seven strains that invaded HTB-9 were from different phylogenetic groups. For the model uropathogenic group B2 strain UTI89, which expresses pili over flagella, loss of flagella or pili impacted invasion into CRL2169 to similar extents, but loss of pili had a greater effect on invasion into HTB-9 and a murine infection model than loss of flagella. A hyperflagellated variant of a group A strain did not invade either cell line better than the parental strain. Reported transcript differences, which were confirmed experimentally, showed that CRL2169 was more differentiated. The endocytosis stimulator tanshinone enhanced invasion into HTB-9, but not into CRL2169, which suggests differences in endocytic pathways and is consistent with differences in differentiation states. If the initial or recurring event in urinary tract infection is invasion into differentiated urothelial cells, as opposed to tight junctions, then the role of flagella may have been underestimated.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718932v1?rss=1

Hogins, J., Nguyen, J., Khuwaja, W., Hall, S., Fogg, V., Dong, X., Zimmern, P. E., Reitzer, L.

Immune receptor LILRB1 mediates cis-signalling which is targeted by RIFINs of the malaria parasite

The malaria parasite, Plasmodium falciparum, replicates within human erythrocytes, where it is susceptible to clearance by immune cells. It uses erythrocyte surface proteins, the RIFINs, to signal through immune receptors and to suppress immune cell function. Previous studies identified two groups of RIFINs which bind different sites on inhibitory immune receptor LILRB1. While some RIFINs bind an elongated LILRB1 conformation, triggering inhibitory immune signalling in trans, we show that other RIFINs stabilise a c-shaped LILRB1 conformation. This buckled LILRB1 binds MHC class I found on the same immune cell, which triggers inhibitory cis signalling. Therefore, LILRB1 exists in dynamic equilibrium, with an elongated conformation able to bind to ligands in trans on a target cell, while a buckled conformation signals through MHC class I in cis, setting the signalling threshold. Different clades of RIFINs exist to mediate inhibitory signalling through each of these LILRB1 conformations to prevent parasite destruction.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718981v1?rss=1

Chamberlain, S. G., Widdess, M., Morch, A., Sakoguchi, A., Sakuno, R., Kurz, E., Chen, L., Valvo, S., Iwanaga, S., Dustin, M., Higgins, M. K.

TIM3+ Tumor Associated M2 Macrophages Impair Antitumor T Cell Immunity and Promote Gastric Cancer Progression and Peritoneal Metastasis

Peritoneal metastases (PM) are the leading cause of cancer-related death in gastric cancer (GC) patients with survival typically < 9 months. Here, we demonstrate that TIM3 and its ligands are increased along the GC continuum and associated with poor survival. Integrated omics analyses and functional studies revealed highly enriched TIM3 in CD163+ tumor associated M2 immunosuppressive macrophages significantly promote tumor cell invasion and tumor growth in vivo, while TIM3 depletion in macrophages reduced tumor cell malignant attributes and increased T cell immunity from PBMCs or CD45+ immune cells of malignant ascites in co-culture system. By cytokine and kinase arrays, we discovered that depletion of TIM3 in macrophages reduced the production of notable secretome of cytokines/chemokines from M2 macrophages; and the protumor function of TIM3+ macrophages rely on the p90RSK1/2/CCL20 axis. Finally, we reveal that TIM3 blockage or genetic KO had superior antitumor activity in combination with anti-PD1 immunotherapy and mitomycin C (MMC) chemotherapy. Together, this study uncovers an important role for TIM3 in tumor associated M2 macrophages and underscores the potential of TIM3 blockage in GC patients with PM.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.716939v1?rss=1

Yao, X., Fan, Y., Zhao, J., Zhang, Y.-t. Y., Athavale, D., Balch, C., Ghelfi, M., Pompetti, A., Zhao, J., Scott, A., Jin, J., Hong, Y. K., Morrison, J., Torres, M., Dhar, S. S., Wang, L., So, J. B.-Y., Tan, P., Sundar, R., Spitz, F., Grana, G., Ajani, J. A., Song, S.

Infra-slow brain-heart-gut electrophysiological interactions reveal a coordinated multisystem physiological network in humans

Growing evidence indicates that brain continuously interacts with other physiological systems through neural and non-neural pathways. The brain-heart and brain-gut axes play a central role in homeostasis, allostasis and behaviour, but also in cognitive aspects including emotion and decision-making. Disruptions in these axes have been linked to a wide range of cardiovascular, neurological, and psychiatric disorders. Despite this evidence, triadic crosstalk between the brain, heart, and gut remains largely unexplored. Brain activity, cardiac autonomic fluctuations, and gastric rhythms all exhibit slow temporal components in resting state, suggesting that brain-heart-gut electrophysiological interactions may occur over timescales from the infra-slow (0.01-0.1 Hz) physiological range. Using non-invasive electrophysiological recordings from 28 healthy participants at rest, we extracted time-varying power dynamics describing the activity of the three organs: brain alpha power, cardiac sympathetic and parasympathetic indices, and the power of the gastric rhythm. Statistical associations among these organs were quantified using the maximal information coefficient across the extended temporal delay range. Physiological interactions were confirmed using surrogate-based testing, which allowed us to construct the network topology of interactions between the three organs. Our findings show that triadic brain-heart-gut interactions form a multi-directional network at infra-slow timescales, shaping resting state activity. This study offers one of the first insights into the physiology of brain-heart-gut interplay, providing a methodological baseline for the development of more comprehensive biomarkers based on network dynamics capable of linking pathological conditions to dysregulation across multiple organ systems.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718683v1?rss=1

Sitti, G., Pitti, L., Candia-Rivera, D.

A cinnamyl alcohol dehydrogenase scaffold organizes monoterpenoid indole alkaloid biosynthesis

Biosynthesis of ~3000 monoterpenoid indole alkaloids (MIAs) including the anticancer drug vinblastine involve the highly unstable intermediate strictosidine aglycone. Its formation by strictosidine {beta}-glucosidase (SGD) and subsequent conversion by geissoschizine synthase (GS) occur in spatially separated compartments, representing a major biosynthesis bottleneck. Here we discover VinBLAST, a cinnamyl alcohol dehydrogenase repurposed as a scaffold for efficient processing of this labile intermediate. VinBLAST physically mediates SGD and GS interaction in the nucleus and allosterically enhances GS catalytic efficiency. VinBLAST homologs from diverse plant families enhance biosynthesis of several representative MIAs, with the production of catharanthine increased to ~160 mg l-1 in yeast, nearly 1000-fold higher than previous studies. Our discovery provides the missing link in organizing MIA biosynthesis and enables scalable bioproduction of geissoschizine-derived therapeutics.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718883v1?rss=1

Gao, D., Mann, S. G. A., Chen, B., Gou, Y., Chen, C., Garza-Garcia, J. J. O., Shahsavarani, M., Jiang, X., Tran, H. C., Bao, J., Richardson, M. B., Li, J., Perley, J. O., Hwang, J., Dong, F., Dong, C., Huang, L., De Luca, V., Wang, Y., Qu, Y., Lian, J.

Expanding the genetic code with diverse backbone structures across diverse sequence contexts

Expanding the genetic code to enable the selective and specific incorporation of non-canonical monomers (ncMs), beyond -L amino acids with variant sidechains, is a key outstanding challenge. Here we discover orthogonal aminoacyl-tRNA synthetases that selectively and specifically acylate their cognate orthogonal tRNA in vivo with eleven new ncMs spanning five different chemical classes: ,-disubstituted-amino acids, malonic acids, carboxylic acids, {beta}2-amino acids and N-cyclic amino acids. We demonstrate that co-translational incorporation of ,-disubstituted-amino acids, {beta}2-amino acids, {beta}3-amino acids and N-cyclic amino acids is strongly dependent on the codons either side of the codon used to direct ncM incorporation, with several ncMs incorporated at less than 1% of sequence contexts. We evolve orthogonal tRNAs that enable the incorporation of previously unincorporated ncMs, enable the incorporation of ncMs at >95% of sequence contexts and, increase the incorporation efficiency at challenging sequence contexts up to 40-fold. We demonstrate the encoded cellular synthesis of proteins and macrocycles containing ncMs and, explicitly demonstrate that our evolved tRNAs provide direct access to a wider range of genetically encoded macrocyclic sequences containing ncMs. Our results provide a foundation for composing, discovering and manufacturing proteins and peptides with functions augmented by ncMs.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718949v1?rss=1

Piedrafita, C., Dickson, A., Richter, D., Weber, C., Elliott, T. S., Liu, Z., Zhang, F., Li, Y., Dunkelmann, D. L., Morgan, T., Liu, K. C., Chin, J. W.

Ionic strength modulates structural disorder and protein oligomerization in the marginally disordered Phd transcription factor

Some proteins combine sequence features that are typical both for folded proteins and intrinsically disordered proteins (IDPs). The borderline properties of these so-called "marginal" IDPs render their conformational ensembles highly sensitive to the environmental changes, which may be important for their function. Here, we investigate the prokaryotic transcription factor Phd, which regulates the phd-doc toxin-antitoxin module through an allosteric mechanism involving disorder-order transition. Using an ensemble of biophysical techniques, we show that the protein is completely disordered at low ionic strength, whereas increasing salt concentration promotes its collapse into a partially ordered monomeric state, followed by the formation of a structured dimer. Using a thermodynamic model, we decipher the linkage between ionic strength, protein stability, oligomer state and degree of disorder. Via small-angle X-ray scattering we derive the structural ensemble of dimeric Phd, revealing a gradation of disorder as a function of salt. The sequence and biophysical properties of Phd position it at the boundary between macroscopically distinct conformational ensembles, representing a large pool of states capable of engaging in functional disorder-to-order interactions, enabling Phd to act as conformational rheostat. Together with previous crystallographic data, this charts the full spectrum of disorder-to-order states in the bacterial transcription factor and underscores the structural plasticity of IDPs with the marginal sequence properties.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718675v1?rss=1

Zavrtanik, U., Muruganandam, G., Prolic-Kalinsek, M., Hammerschmid, D., Sobott, F., Volkov, A. N., Loris, R., Hadzi, S.

Benchmarking Tools for Identification of rRNA Modifications in Escherichia coli using Oxford Nanopore Direct RNA Sequencing

RNA modifications are important for RNA structure, stability, and ribosome function, but their identification and localisation remains challenging. Oxford Nanopore direct RNA sequencing (DRS) enables modification-agnostic detection in native RNA, but existing tool benchmarks have focused almost exclusively on m6A in eukaryotic mRNA, leaving multi-modification tool performance in bacterial systems largely untested. Here, we benchmark ten RNA modification detection tools spanning signal-comparison, error-rate, and hybrid approaches on Escherichia coli K-12 MG1655 16S and 23S rRNA, which harbour 11 and 25 known modified sites, respectively, across 17 modification types. Using native RNA and in vitro transcribed (IVT) unmodified RNA, we evaluate performance across 25 coverage levels (5x to 1000x). DiffErr and JACUSA2 showed the strongest discrimination performance (AUROC >0.9 on both 16S and 23S rRNA), with DiffErr achieving the highest F1 score on 16S and JACUSA2 showing the most consistent precision-recall balance across both rRNAs. Both tools achieved full transcript-wide scoring and, along with DRUMMER, exact positional localisation. Several other tools produced no output at many rRNA positions, and restricting evaluation to reported positions inflated apparent performance. Signal-based tools showed a systematic 1-4 nucleotide 5'; offset from known modified positions, consistent with the ~5-mer nucleotide stretch present in the read head of the nanopore; applying tool-specific offset corrections substantially improved per-site recovery and reduced false positives, substantially improving the performance of tools such as EpiNano and nanoDoc. At single-site resolution, no known modified site was recovered by all tools, and several m5C, m5U, and m6A sites were missed by the majority of tools. Tool combination analysis showed that pairing error-rate-based tools with offset-corrected signal-based tools improved site recovery beyond any individual tool, with the best three-tool combination recovering 30 of the 36 known sites while maintaining low false positive rates. These results establish that discrimination metrics (e.g. AUROC) alone are insufficient to evaluate modification detection tools: output completeness, positional precision, and per-modification-type sensitivity should be reported alongside standard benchmarking metrics.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718756v1?rss=1

Morampalli, B. R., Silander, O. K.

Native entanglement misfolding contributes to age-associated structural changes across the Saccharomyces cerevisiae proteome

Aging at the subcellular level involves the simultaneous decline in the cell's ability to maintain protein homeostasis and rise in misfolded proteins through a positive feedback loop. Here, we test if a widespread class of protein misfolding could contribute to proteome aging by examining if statistical associations exist between age-related changes in protein structure, measured by limited proteolysis mass spectrometry data of the aging Saccharomyces cerevisiae proteome, with structural annotations and molecular simulations. We find that globular proteins that are likely to exhibit entanglement misfolding are 121% more likely to exhibit age-related structural changes, and these changes are 59% more likely to be localized to natively entangled regions. Proteins containing native entanglements are seven-fold more likely to misfold, according to simulations, and populate long-lived, near-native misfolded states. Thus, the age-related structural changes in yeast proteins can be explained in part by the accumulation of misfolded proteins involving entanglements.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.717356v1?rss=1

Vu, Q. V., Sitarik, I., Nissley, D. A., O'Brien, E. P.

The Effects of Phosphorylation on the Structure and Function of Motif A, an Intrinsically Disordered Region within SIRT1

The NAD+ dependent deacetylase sirtuin-1 (SIRT1) is known to elicit cellular defenses against aging, cancer, and other aberrant pathologies. Previous studies have identified an intrinsically disordered region of SIRT1 comprised of N-terminal residues 1-52, herein referred to as motif A, which activates SIRT1 activity, likely through intramolecular interactions. Additionally, phosphorylation of N-terminal residues Ser27 and Ser47 has been shown to be important for regulating SIRT1 activity and stability. The lack of in vitro characterization of these effects hampers our further understanding of the role of motif A in SIRT1 regulation. In this study, we elucidate the role phosphorylation plays in motif A structure as well as its regulatory effects on SIRT1 activity against Ac-p65. We find that phosphomimetic mutation at Ser27 significantly increases the activation effect of motif A towards SIRT1. This result is supported by molecular dynamics simulations of the phosphomimetics, which reveal stabilization of different transient structures for motif A depending on whether Ser27 and Ser47 have been modified. A key finding suggested by this study is that phosphorylation of S27 appears to activate SIRT1 by causing motif A, which is intrinsically disordered in the WT, to fold into an ordered structure. This conclusion is based on both the experimental findings and simulation results. These findings contribute to our understanding of SIRT1 regulation, specifically the role played by phosphorylation within the N-terminal disordered region.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718858v1?rss=1

Richter, S. M., Bui, H.-L., Chen, A., Tannous, C., Butler, B. R., Bennett, S. D., Nguyen, S. Q.-a., Prado, J., Mohamed, A., DuBois, I. A., Tadros, E., Thai, N. T., Lima Guan, S., Peralta, C. M., Kwong, A., Hawk, L. M. L., Grazioli, G., Wang, N.

Genome sequencing and multi-stage, blood-feeding, and tissue-specific transcriptome atlas of the Rocky Mountain wood tick provide a critical resource for this vector

Dermacentor andersoni, the Rocky Mountain wood tick, is an important vector for pathogens impacting human and animal health, including bovine anaplasmosis, Colorado tick fever, and Rocky Mountain spotted fever. A better understanding of the biology of this tick is needed for developing disease prevention and vector control strategies. A reference genome was assembled for D. andersoni using high-fidelity (HiFi) long-read PacBio sequences and HiC contact mapping, yielding a contiguous assembly in which most contigs matched one of 11 chromosomes. Genome annotation by the NCBI eukaryotic genome annotation pipeline revealed high gene content completeness, yielding a genome completeness score of 94.0% using the Arachnida ortholog dataset. Following genome sequencing, we identified specific genes involved in blood feeding across a range of tissue types and life stages for D. andersoni. To accomplish this, RNA-seq analysis was used to investigate differential gene expression across most organs in adult, nymphal, and larval D. andersoni before and after feeding. Based on this analysis, we identified several gene groups that are involved in blood feeding. Furthermore, we establish sex- and developmental-stage-specific transcriptional profiles. Collectively, this study advances knowledge of D. andersoni biology and enables the development of strategies to limit the spread of diseases transmitted by this tick.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.717773v1?rss=1

Tompkin, J. E., Saelao, P., Kruczalak, J., Yeo, H., Olafson, P. U., Sim, S. B., Oyen, K., Kelley, M., Corpuz, R. L., Scheffler, B., Geib, S. M., Childers, A., Chen, X., Weirauch, M. T., Dergousoff, S. J., Soghigian, J., Noh, S. M., Benoit, J.

Expansion and Differentiation of Adult Human Pancreas-Derived Progenitor Cells into Functional Islet-Like Organoids

Background and Aims: Adult pancreas-derived islet progenitor cells (IPCs) have recently been shown to expand in culture and differentiate into endocrine-like organoids. However, translation of this approach to a clinically compatible workflow requires cell enrichment strategies and validation using tissue obtained during real-world clinical procedures. Here, we adapted our previously described IPC platform to non-endocrine pancreatic tissue fractions generated during clinical islet isolation procedures and evaluated their capacity to generate functional islet organoids. Methods: Non-endocrine pancreatic tissue fractions obtained during clinical islet isolation were expanded ex vivo and enriched using fluorescence-activated cell sorting (FACS) for CD81 and CD9, surface markers previously identified in IPC populations. Sorted cells were expanded, induced to form IPC clusters, and differentiated with ISX9 to generate islet organoids. Differentiation was assessed by gene expression analysis, flow cytometry, immunofluorescence, calcium flux assays, glucose-stimulated insulin and glucagon secretion, and single-cell RNA sequencing. Results: Clinically derived non-endocrine cell fractions yielded expandable IPC populations expressing progenitor-associated markers. FACS-purified and expanded CD81+/CD9+ IPCs were enriched with BMPR1A and P2RY1. Sorted cells generated three-dimensional BMPR1A+ and RGS16+ IPC clusters. IPC clusters differentiated into islet organoids with upregulated expression of canonical beta- and alpha-cell transcription factors. Single-cell transcriptomic profiling revealed activation of coordinated endocrine gene programs and alignment with reference human islet endocrine signatures, while the undifferentiated IPC compartment was marked by enrichment of PTX3, FST, CEMIP, and GREM1. Terminally differentiated cells exhibited depolarization-induced calcium influx and glucose-regulated insulin and glucagon secretion. Conclusions: These findings establish an adaptable workflow for expansion and production of functional islet organoids recovered from clinically derived pancreatic tissue. This strategy may provide an unlimited autologous source of adult progenitor-derived islets for future islet cell replacement therapies in diabetes.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.14.718604v1?rss=1

Kuncha, J., Darden, C. M., Kirkland, J. T., Blanck, J.-P., Fowlds, K., Cho, M., Danobeitia, J. S., Naziruddin, B., Lawrence, M. C.

In silico structural analysis of EthA substitutions for ranking priority mutations leading to ethionamide resistance in Mycobacterium tuberculosis

Background: Tuberculosis (TB) is the second-leading cause of deaths from infectious agents and remains a global health threat. Ethionamide (ETH) is a prodrug used in regimens for multidrug-resistant TB, and, partly due to side effects that can lead to low treatment adhesion, resistance arises. Changes in EthA, the monooxygenase that activates ETH, are the main mechanism of resistance. Yet, of hundreds of EthA substitutions found in resistant isolates, only a handful have been annotated as resistance determinants. Results: An in silico analysis was carried out on a previously described panel of Mycobacterium tuberculosis clinical isolates for which genomes and ETH susceptibility testing results were available. EthA substitutions were mapped, revealing the existence of hotspots in its sequence. Visualization of the hotspots in the EthA structural model shows that they cluster in three regions, including ligand binding pockets. Models were built of twenty-three variants found in resistant isolates and changes in local configuration was mapped to identify investigate impact on ETH activation. Information from these models contributed to establishing five criteria for scoring whether substitutions are most likely to lead to resistance. Using these criteria, EthA D58G was selected and its expression is shown to increase growth in high ETH concentrations. Conclusion: Functionally relevant regions of EthA are revealed and point out priority substitutions for functional studies, enhancing identification and detection of substitutions not been previously associated with resistance.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718980v1?rss=1

Machado, R. F., Cardoso, S. L., Pinheiro, I. C., Ramos, J. P., Antunes, C., Capriles, P., Galvao, T. C.

Mosquito-directed PROTACs to block malaria transmission

The mosquito stage of the Plasmodium falciparum life cycle is an attractive target for intervention since it is crucial for the sexual reproduction and transmission of parasites to human host. Mosquito determinants crucial for parasite infection and growth pose as lucrative targets for transmission blockers. Owing to the fact that p38 MAPK has role in immune response and vector competence, we have evaluated the potential of PROTAC molecule (NR-7h) to degrade Anopheles stephensi p38 MAPK (Asp38 MAPK), a conserved serine/threonine kinase involved in stress reactions, midgut homeostasis, and parasite survival. PROTAC-mediated degradation of Asp38 MAPK led to the disrupted development of the parasite, suggesting its crucial function in vector competence. Furthermore, NR-7h-treated mosquitoes showed higher expression of immune genes such Rel-2, TEP1, APL1, and NOS, suggesting that p38 MAPK regulates host immunity in a way that promotes parasite persistence. PROTAC-mediated degradation of target proteins, provides a more persistent and resistance-proof therapeutic effect than traditional kinase inhibitors. Our findings establish PROTACs as a novel vector-targeted strategy for the development of endectocides to limit malaria transmission.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.719026v1?rss=1

Rawat, N., Singhal, J., Goyal, B., Parveen, N., Tupe, C., Gupta, K., Chakraborti, S., Pandey, K. C., singh, s.

Chromatin association promotes UBR5-mediated degradation of Rb

The retinoblastoma protein Rb is a cell cycle inhibitor that plays a central role in regulating the G1/S cell cycle transition. Un-/hypo-phosphorylated Rb suppresses E2F transcription activity by binding to E2F/DP dimers and recruiting chromatin remodelers to prevent cells from entering S phase. For cells to progress through the G1/S transition, Rb is inactivated by two mechanisms: the classic pathway of Rb hyperphosphorylation by Cyclin-CDK complexes, and a recently identified degradation mechanism driven by the E3 ubiquitin ligase UBR5. These two pathways are interconnected, as only the un-/hypo-phosphorylated Rb can be degraded, and the hyper-phosphorylated Rb is stabilized to promote its reaccumulation in preparation for the next cell division cycle. However, the molecular basis for how Rb is stabilized upon phosphorylation remains unclear. In this study, we found that UBR5 preferentially targets chromatin-associated proteins for degradation. Since the chromatin association of Rb is modulated by its phosphorylation, we hypothesized that phosphorylation may affect Rb stability by altering its chromatin association. To test this, we constructed a series of un-phosphorylatable Rb variants with graded reductions in chromatin association. Consistent with our hypothesis, we observed a strong correlation between the chromatin association of a Rb variant and its half-life. Fusing these Rb variants to histone H1 increased chromatin association to similar levels and equalized their protein half-lives. Taken together, these findings show how phosphorylation stabilizes Rb by promoting its dissociation from chromatin. This provides a striking example for how sub-organellar protein localization may be used to regulate stability.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.719064v1?rss=1

Zhang, S., Lanz, M., Konschnik, J., Skotheim, J.

Evolution of the highest fidelity DNA replication systems

DNA mutation on average is deleterious, and evolution generally acts to reduce mutation rates to the limit of natural selection. The limit of natural selection is set by multiple factors, of which effective population size is only one. We consider a form of lethal mutagenesis as an upper bound to mutation rates for any organism, an argument that is congruent with a biophysical context, wherein random mutations are a form of entropy. In this analysis, coding genome size, body mass, generation time, and temperature explain more than 90% of the variation in mutation rate per generation across the Tree of Life. The organisms with larger genomes, longer lifespans and relatively larger body sizes, known and unknown, represent the lineages which have likely evolved novel mechanisms to lower mutation rates. Though these variables are largely shared by Peto's Paradox, this selective pressure occurs through germline mutation rate evolution rather than the soma.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.719065v1?rss=1

Baehr, S., Call, C.

An integrated pipeline to count individual transcripts with single-cell resolution

Quantifying transcript abundance at single-cell resolution is important for understanding gene regulation in intact multicellular organisms. In Caenorhabditis elegans, RNA fluorescence in situ hybridization has been widely used to visualize transcripts, but conventional smFISH approaches can be limited by low signal-to-noise, poor performance with short transcripts, and workflows that do not readily support absolute transcript counting in identified cells. Here, we present an integrated experimental and computational pipeline for quantitative transcript analysis in whole-mount C. elegans embryos, larvae, and adults. The pipeline combines Hybridization Chain Reaction (HCR), confocal microscopy, RS-FISH spot detection, manual cell annotation in FIJI, and custom MATLAB-based spot assignment to quantify individual transcripts within defined cells. We show that this approach enables sensitive, specific, and multiplexed detection of transcripts, including short insulin-like peptide mRNAs, with single-cell resolution. Known spatial expression patterns were resolved in embryos, larvae, and adults, and probe specificity was validated using deletion mutants that remove HCR probe-binding sites. Applying this pipeline to ins-4, ins-6, and daf-28 in ASI and ASJ sensory neurons revealed cell-specific and context-dependent regulatory relationships across multiple mutant backgrounds. This workflow provides an accessible method for absolute transcript counting in anatomically intact C. elegans and should support mechanistic studies of gene regulation, cellular heterogeneity, and transcriptional network function.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.15.718387v1?rss=1

Sheardown, E., Yan To Ling, J., van der Burght, S. N., Vaikkinen, H., Gowing, B., Ahringer, J., Hamid, F., Ch'ng, Q.

Metatranscriptome data support the existence of two distinct morphotypes in a single parmalean species in natural environments

Parmales (Bolidophyceae), the closest relatives of diatoms, includes isolates solely exhibiting one of two distinct morphotypes: a silicified non-flagellated form (S-type) and a naked flagellated form (F-type). Although alternation of these two forms for a single isolate has not been observed, previous studies hypothesized that these morphotypes represent alternating stages of a single parmalean. In this study, we investigated the global expression patterns of S- and F-type marker genes by integrating parmalean Metagenome-Assembled Genomes (MAGs) and the metatranscriptomic dataset from Tara Oceans. We detected the expression of both S- and F-type marker genes from individual environmental MAGs. This finding provides the first metatranscriptomic evidence that natural parmalean genomes possess the potential to manifest both morphotypes. Furthermore, our analysis revealed different geographical expression patterns between the two forms. The expression of F-type markers showed a broad distribution, whereas that of S-type markers was more restricted, suggesting distinct niches for the two morpho-phases. Moreover, S-type gene expression appears to require specific environmental triggers that lead to a higher population density, whereas F-type expression is rather constitutively maintained. Overall, our results support the hypothesis of a life cycle involving morphological switching and reconcile the long-standing discrepancy between the ubiquity of parmaleans in molecular surveys and the limited geographical range for the observation of silicified cells. Based on these patterns, we propose a threshold-based model in which F-type-dominated populations persist under conditions unfavorable for growth and a morphological switch to the S-type is triggered once environmental conditions exceed a critical threshold for growth.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718586v1?rss=1

Sasaki, H., Endo, H., Pelletier, E., Yoshikawa, S., Kuwata, A., Ogata, H.

Functional Genomics Reveals TNT Bioremediation Strategies in Pantoea sp. MT58 and Pseudomonas putida KT2440

2,4,6-Trinitrotoluene (TNT) is a recalcitrant and pervasive environmental pollutant. Although different environmental microbes have demonstrated their ability to degrade or transform TNT, the underlying genetic basis and cellular machinery remain unclear. In this study, we investigated bacterial strategies in response to TNT exposure in Pantoea sp. MT58 and P. putida KT2440 using proteomics and random barcode transposon-site sequencing (RB-TnSeq). Pantoea sp. MT58 was found to utilize TNT as a sole nitrogen source, whereas P. putida KT2440 exhibited only stress tolerance without assimilation. Pantoea sp. MT58 encodes multiple putative nitroreductases that were upregulated, yet deletion of these genes did not affect growth on TNT, revealing pathway redundancy. Furthermore, fitness profiling provided no evidence for genes involved in the canonical Meisenheimer-complex pathway associated with nitrite release. Instead, the data are most consistent with a sequential nitro-group reduction route in which nitrogen is ultimately recovered as ammonium, with nitrogen routed through the GS-GOGAT pathway with purine and urea pools as the candidate buffering architecture for TNT mineralization. Conversely, P. putida KT2440 relied on Ttg/RND efflux pumps and toluene tolerance proteins for survival without nitrogen assimilation from TNT. This work distinguishes routes for productive nitrogen assimilation from those involved in nitroaromatic tolerance, expanding the mechanistic understanding of anthropogenic compound metabolism to inform future bioremediation efforts.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.711451v1?rss=1

Wang, L.-W., Eng, T., Rivier, A., Naseem, S., Codik, A., Chen, Y., Srinivasan, A., Petzold, C. J., Nelson, K. L., Deutschbauer, A. M., Mukhopadhyay, A.

Environmental DNA reveals long-term persistence of a Midichloria-like bacterium in a rainbow trout aquaculture and links Ichthyopthirius multifiliis with the red mark syndrome

Red Mark Syndrome (RMS) is a widespread skin disease affecting rainbow trout (Onchorhynchus mykiss). It provokes substantial economic losses in aquaculture, and is putatively caused by a Rickettsiales bacterium named Midichloria-like organism (RMS-MLO), which is strongly associated with RMS lesions. However, RMS-MLO ecology and epidemiology in aquaculture systems remain poorly understood. In this study, we analysed environmental DNA to monitor the presence of RMS-MLO and its putative vector Ichthyophthirius multifiliis in a trout farm in Northern Italy over one year. Water and sediment samples were monthly collected from multiple water tanks. RMS-MLO was consistently detected by PCR throughout the study in all trout-containing tanks, both in water and sediment samples, but never in the trout-free inflow tank. We did not observe an increase in RMS-MLO abundance during the single RMS outbreak recorded nor in relation with the co-occurrence of I. multifiliis. Our findings indicate a long-term persistence of RMS-MLO in the aquaculture, possibly as a consequence of infections with low prevalence or abundance, rather than its entry from the external environment at the time of RMS outbreaks. Additionally, hints were recorded for a potential role of free-living aquatic microeukaryotes as additional occasional reservoirs. In contrast, I. multifiliis was negatively related with RMS-MLO, while it significantly increased in abundance during the RMS outbreak, particularly in the inflow tank. This supports that, rather than a stable reservoir, I. multifiliis may act as a facilitator of RMS outbreaks, which might indeed be triggered by the entry of this parasite in trout farms.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.718929v1?rss=1

Vecchio, D., Siviglia, Y., Allievi, A., Fesce, E., Losi, P., Croci, C., Gammuto, L., Ilahiane, L., Melis, S., Cafiso, A., Ferrari, N., Petroni, G., Serra, V., Tedesco, P., Castelli, M.

Dynamic cancer dormancy and awakening emerge from tumor microenvironment feedback in a minimal theoretical model

Cancer cell dormancy is characterized by late relapse and therapy resistance, yet the mechanisms that awaken dormant cells remain poorly understood. The tumor microenvironment has emerged as a key driver of these state transitions. Here we present a theoretical framework based on evolutionary game theory in which interactions between cancer and host cells are coupled explicitly to a changing tumor microenvironment. Cancer cells produce a conditioning factor that is cleared by the microenvironment and tolerated only up to a threshold. Through this conditioning factor, the microenvironment feeds back on cancer-host interactions and reshapes their competitive balance. Unlike a model with fixed interactions, this feedback allows dormancy and awakening to emerge as dynamic outcomes of microenvironmental change. We show that this minimal coupling is sufficient to generate distinct long-term regimes. Across these regimes, feedback generates thresholds and history dependence, so that the same cancer population can follow different fates depending on whether the microenvironment is already primed. Our framework reduces these dynamics to experimentally and biologically interpretable parameters linked to conditioning factor production, clearance, tolerance, and microenvironment-dependent changes in cancer-host competition. More broadly, it provides a quantitative basis for testing how collective microenvironmental feedback shapes cancer dormancy and awakening, and for designing experiments to uncover the mechanisms that awaken dormant cancer cells.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.14.718509v1?rss=1

Yanez Feliu, G. A., Rossato, G., Valleriani, A., Cipitria, A.

ADAR-Sense: an open-access, species-agnostic web tool for automated, user-customisable ADAR-based RNA sensor design

Background: Engineered synthetic RNAs enable cellular control by sensing and responding to intracellular biomolecules. Recently developed sense-edit-switch RNAs (sesRNAs) based on Adenosine Deaminase Acting on RNA (ADAR)--which edits a stop codon to switch on custom payload translation in the presence of a target RNA--are consistently functional across different species. The ability of sesRNAs to couple bespoke payload translation to the presence of cell type-specific transcripts will usher in an era of precise cell-targeted biotechnological interventions. Results: To expedite the generation of sesRNAs, we develop ADAR-Sense--a universal web tool for automated sensor design based on user-defined sensor length, sensor-target RNA mismatch number, mismatch proximity to the ADAR-editable stop codon, and targeted custom element inclusion for improved ADAR recruitment and subsequent payload induction. Conclusions: Compared to current tools, the simplicity and flexibility of ADAR-Sense will streamline the design and screening of sesRNAs in new cell types and conditions, supporting the swift adoption of this sensing platform in both basic and translational research.

Date: 2026-04-17
https://www.biorxiv.org/content/10.64898/2026.04.16.719097v1?rss=1

Sserwadda, H., Park, K., Kim, Y.-H., Kim, H. J., Park, C.-G.