Biology subjects

Williams, T.

Publications and source records attributed to Williams, T..

At least 19 recordsLinked to original sources

bioRxiv2026

NEIL2 regulates RNA polymerase II dynamics to suppress transcription replication conflicts in pancreatic cancer

Transcription replication conflicts (TRCs) arise from spatiotemporal interference between replication and transcription machinery and contribute to genome instability in cancer cells. In pancreatic ductal adenocarcinoma (PDAC) cells, oncogenic KRAS drives TRC formation, and base excision repair (BER) contributes to TRC resolution as an adaptive response. Here, we show that TRCs are enriched in pancreatic tumor sections from patients and are associated with malignant progression. We identify NEIL2, a bifunctional DNA glycosylase in the BER pathway, as a critical regulator of TRCs in PDAC cells. Mechanistically, NEIL2 loss causes aberrant RNA polymerase II (RNAPII) occupancy at transcription start sites and downstream of transcription end sites. Dysregulated RNAPII dynamics promote R-loop-dependent and -independent TRCs contributing to replication stress, DNA damage, and impaired cancer cell proliferation. Notably, the DNA-binding motif of NEIL2 necessary for canonical BER function is dispensable for TRC regulation. Together, these findings reveal an unrecognized role for NEIL2 in regulating RNAPII dynamics and mitigating TRCs in pancreatic cancer cells.

molecular biology↗

bioRxiv2026

Robust V(D)J Recombination in the Absence of SA1/2-mediated Chromatin Loops

The cohesin ring complex organizes and mediates functional activity of the genome through chromatin loop extrusion1,2. Cohesin comprises a core SMC1-SMC3-RAD21 ring associated with multiple regulatory proteins, including SA1 and SA2 paralogs that bind mutually-exclusively to a RAD21 site3,4. Functional studies of cohesin complexes in vivo often employ inactivation of particular components2,3. However, inactivation of SA1 or SA2 in mice is limited by mouse embryonic lethality5-7 and, for dual SA1/2-inactivation by cycling cell lethality8-10. For these reasons, it remains unknown whether the requirement for SA1/2 in loop extrusion in vitro11,12 applies in vivo1,2, and whether these factors have overlapping or distinct functions in regulating this process3. Here, we employ G0/G1-arrested v-Abl pro-B lines, which are viable in the absence of cohesin-complex factors13,14, to dissect SA1 and SA2 functions in V(D)J recombination, a strictly G0/G1-specific process that harnesses loop extrusion to effect distinct Igh and Ig{kappa} locus recombination mechanisms15,16. Robust long-range Igh or Ig{kappa} V(D)J recombination continued unabated in the absence of SA1 or SA2. However, inactivation of both abrogated most positioned chromatin loops genome-wide and also abolished long-range Igh V(D)J recombination with a phenotype similar to RAD21 (cohesin) inactivation13. Yet, robust Ig{kappa} V(D)J recombination continued upon dual SA1/2-inactivation, which along with Hi-C findings, demonstrates ongoing loop-extrusion in the absence of SA1/2. Correspondingly, replacement of weak Igh V(D)J recombination signal sequences (RSSs) that target V(D)J recombination activity, with strong Ig{kappa} counterparts15 restored long-range Igh V(D)J recombination in SA1/2 inactivated cells. We conclude that SA1/2 together maintain loop extrusion impediments and genome-wide architecture in G0/G1-arrested cells; and, in their absence, loop extrusion proceeds unimpeded. Remarkably, strong RSSs allow Ig{kappa} to function normally in the absence of internal positioned loop structure across the 3.1-mega-base Ig{kappa} locus. We discuss implications of our findings for long-range gene regulation in G0/G1-arrested cells more broadly.

immunology↗

bioRxiv2026

Sediment depth-dependent trait expression controls species contributions to nutrient cycling

Global biodiversity loss is restructuring ecological communities and compromising ecosystem functioning. Yet, we still lack an empirical understanding of how specific trait combinations regulate ecosystem processes or how their functional expression is organised within the immediate environment. Here, we investigated trait-expression across 17 functionally contrasting sediment-dwelling marine invertebrate species. We show that while sediment reworking and burrow ventilation are depth dependent, their influence on nutrient concentrations are highly nutrient-specific. Our analyses reveal that nutrient concentrations are better explained by continuous, spatially explicit trait combinations than by categorical bioturbation modes. Surface modification was associated with higher ammonium concentrations, moderated by ventilation, while deeper sediment reworking and biomass were associated with higher nitrite concentrations. Phosphate relationships were weak, and nitrate was not explained by the measured traits. These results establish a mechanistic baseline for species-specific trait-nutrient relationships under controlled conditions. This shift from categorical trait assignments to a spatially explicit, empirically resolved trait-interaction framework provides a mechanistic foundation for predicting how changes in species composition may alter benthic nutrient cycling.

ecology↗

bioRxiv2026

Cell cycle dependent regulation of mRNA export in response to replication stress

Nuclear export of mRNA is extensively coupled to transcription and processing of mRNA. How mRNA export is mechanistically regulated remains poorly understood. Here, we uncover a physical checkpoint at nuclear speckles that regulates mRNA export during DNA replication in S-phase of the cell cycle. Unbiased screening approaches identify WEE1 and CHK1 as novel mRNA export regulators. mRNA export complexes are recruited to sites of replication stress in S-phase. WEE1 inhibition prematurely activates CDK1 and PLK1, leading to accumulation of R-loop associated mRNA in large nuclear speckles, with late markers of replication stress present around their periphery. This recruitment is dependent on CDK1 activity. Phosphorylation of ALYREF by CDK1 and PLK1 regulates nuclear speckle accumulation of mRNA following replication stress. mRNA export factors including ALYREF are subsequently mis-localised from these speckles, preventing nuclear export of R-loop associated mRNA. Thus, WEE1, CDK1 and PLK1 enforce a cell cycle regulated checkpoint at nuclear speckles that serves to protect the cell from major sources of genome instability by ensuring that R-loop associated mature mRNA is not exported to the cytoplasm.

molecular biology↗

bioRxiv2026

Tfh dysfunction underlies poor responses to HBV vaccination

The primary goal of vaccination is to induce durable protection through generation of antigen-specific humoral immunity and cellular immune memory. Yet the same vaccine may elicit durable protection in some and less durable responses in others. Antigen-specific antibody responses arise from germinal centers (GCs), where T follicular helper (Tfh) cells help cognate B cells undergo somatic hypermutation (SHM), affinity maturation to generate humoral protection and cellular immune memory. In humans, however, the mechanisms linking GC dynamics to downstream antibody output remain incompletely characterized. To identify the molecular and cellular events that underlie differential vaccine responsiveness, we leveraged the known, pronounced variability in vaccine antigen-specific antibody concentration and durability observed after Hepatitis B virus (HBV) immunization. We longitudinally profiled 101 healthy adults undergoing either de novo (n=59) or booster (n=42) immunization. We then stratified participants into High (>100 mIU/mL) and Low (<100 mIU/mL) responder groups based on vaccine antigen-specific antibody responses. From this cohort, we obtained core needle biopsies (CNBx) of vaccine-draining lymph nodes from 10 participants (High Responder n=6, Low Responder n=4), 10-21 days post-final vaccination and performed single-cell RNA sequencing with paired antigen receptor sequencing. Lymph node BCR repertoire analysis revealed convergent, semi-public clonotypes across unrelated participants with elevated SHM. Transcriptomic profiling of GC-B cells uncovered stark functional divergence between response groups, with High Responders showing robust activation across both dark zone (DZ) and light zone (LZ) compartments and enrichment of MYC and mTORC1 signaling. In contrast, Low Responders exhibited a contracted LZ compartment and a stalled GC B cell response. This divergence in B cell responses corresponded to changes in the T cell compartment. High Responders harbored an expanded Tfh subset marked by increased expression of CXCL13, ICOS, and GNG4, indicative of their GC localization and capacity to provide B cell help. Moreover, receptor-ligand analysis indicated that these GNG4+Tfh formed coordinated costimulatory networks capable of driving LZ GC-B cell licensing. Low Responders demonstrated a divergent program in which their GNG4+Tfh displayed aberrant inflammatory transcriptional activity. This inflammatory signaling was associated with reduced specificity of BCL6 regulon activity despite maintained expression of canonical Tfh genes. Together, these findings reveal that variable HBV vaccine responsiveness was associated with transcriptional network alterations in the GNG4+Tfh subset, offering a molecular framework for understanding poor vaccine responses. One Sentence SummaryLymph node Tfh dysfunction is associated with poor vaccine responses to Hepatitis B virus vaccination

immunology↗

bioRxiv2026

Human antibody heavy chain variable region exon interacts with 3' regulatory region to form a stable somatic hypermutation center

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) of immunoglobulin heavy chain (HC) variable region exons in germinal center (GC) B cells, allowing selection of affinity-matured B cell receptors. The V(D)J exon location is privileged for SHM targeting of diverse sequences within it. In analogy to the related HC class switch recombination center, we proposed that cohesin-mediated loop extrusion juxtaposes widely separated enhancers, including 3 Igh regulatory region ("3RR"), with the V(D)J exon in GC B cells to establish a SHM-center (SHM-C) "privileged" for AID access. To test this hypothesis, we employed a human RAMOS GC B lymphoma cell SHM model with inducible AID expression in which we targeted an unmutated mouse HC V(D)J exon in place of its human counterpart. Activation of AID expression in this cycling RAMOS line for 10 days led to robust SHM accumulation in the mouse V(D)J exon with a pattern similar to that in mouse GC B cells. In this model, the mouse V(D)J exon was indeed juxtaposed to the two human 3RRs and other elements consistent with SHM-C formation. While deletion of individual 3RRs had little effect, deletion of both greatly reduced SHM, but left V(D)J exon transcription unabated. Moreover, the SHM-C and SHM accumulation remained intact over a 10-day period in viably G1-arrested and viably G1-arrested RAD21 (cohesin)-depleted versions of the model. We discuss implications for SHM-C structure and function, its long-term functional stability in the absence of the loop extrusion process proposed to assemble it, and for 3RR SHM functions beyond transcriptional activation. Significance StatementUpon encountering pathogens, B cells enter specialized lymphoid structures known as germinal centers (GCs). GC B cells express activation-induced cytidine deaminase (AID) that initiates somatic hypermutation (SHM) of the portion of antibody genes that encodes antigen recognition. B cells in which mutations increase antibody affinity for activating pathogen are selected to enhance antibody responses. We proposed SHM occurs in a chromosomal structure in the antibody gene locus termed a SHM-center (SHM-C). By studying SHM in a human B cell lymphoma model, we provide direct evidence for existence of a SHM-C assembled from different functional components normally separated by considerable distances. We also find that the SHM-C is surprisingly stable and continues to function for many days in non-dividing cells.

immunology↗

bioRxiv2026

Mapping the global distribution and spread of the Plasmodium vivax-associated virus MaRNAV-1

Matryoshka RNA virus 1 is a bi-segmented and single-stranded RNA virus associated with Plasmodium vivax, a cause of human malaria. Little has been uncovered about the epidemiology and ecology of this virus since its discovery in 2019. To address this, we used a combination of primary and publicly available metatranscriptomic data to map the geographic distribution and host associations of MaRNAV-1. We detected this virus throughout Southeast Asia, in parts of South America, and, for the first time, in Oceania. Despite its broad distribution, MaRNAV-1 was found exclusively in metatranscriptomes containing P. vivax, suggesting that there is a specific virus-host relationship that has shaped the evolutionary history of this virus. We were unable to estimate the emergence date of the MaRNAV-1 lineage; however, phylogeographic mapping analysis suggested that MaRNAV-1 may have radiated from Southeast Asia. Our findings have both evolutionary and public health implications and can serve as the basis for future investigations in these fields.

evolutionary biology↗

bioRxiv2026

Notch signaling in the embryonic ectoderm promotes periderm cell fate and represses mineralization of vibrissa hair follicles

The Notch signaling pathway is a critical means to regulate cell fate choice in animals. Appropriate regulation of this pathway is also required for human face formation as both loss and gain of function mutations of Notch signaling can cause syndromes with craniofacial abnormalities. Here we examine the consequences of manipulation of Notch signaling in the early mouse embryonic ectoderm by either removing the transcriptional effector Rbpj or expressing a constitutively active form of the Notch1 intracellular domain. Loss of Rbpj resulted in cleft secondary palate but strikingly was also associated with the ectopic mineralization of vibrissa follicles. In contrast, activation of Notch signaling resulted in multiple embryonic defects including a fully penetrant bilateral cleft lip and palate. Further, single cell RNA-seq data indicated a switch from a basal epithelial cell identity towards periderm when Notch signal transduction was elevated. These cell fate changes were accompanied by misregulation of genes and pathways known to impact human and mouse orofacial clefting including Grhl3, Irf6, and Wnt pathway. Together, these findings provide insight into human craniofacial conditions caused by misregulated Notch activity. SUMMARY STATEMENTOur studies demonstrate that Notch signaling in the embryonic ectoderm stimulates periderm cell fate while also repressing transformation of the inner root sheath of whisker follicles into mineralized tissue.

developmental biology↗

bioRxiv2025

Protective and pathogenic antibody responses from a primate Shigella outbreak inform vaccine design

There is currently no approved vaccine for Shigella spp., a leading cause of diarrhea that are increasingly resistant to antimicrobials. Shigella vaccine development is complicated in part by an incomplete understanding of the structural and molecular determinants of immunity. To address this, we isolated monoclonal antibodies (mAbs) against candidate Shigella vaccine antigens using samples from a Shigella flexneri outbreak in a non-human primate (NHP) research facility. We found that antibodies targeting the Shigella O-antigen (O-Ag) can undergo significant affinity maturation (>10%) to acquire broad cross-reactivity across S. flexneri serotypes. We also found that the virulence-associated type III secretion system (T3SS) proteins IpaD and IpaB elicit moderate T cell and robust antibody responses. T3SS antibodies either inhibit, or - surprisingly - enhance bacterial virulence in vitro and in vivo depending on their epitope specificity. These findings provide key insights into protective and deleterious immune responses against Shigella that directly inform vaccine immunogen design.

immunology↗

bioRxiv2025

Endothelial-targeted CD39 prevents Toxin-induced Pulmonary Hypertension Mice.

Disruption of the pulmonary endothelium by drugs, toxins, viruses (e.g., COVID-19), or bacterial sepsis can cause acute pulmonary vasculopathy leading to pulmonary hypertension and consequential heart failure. CD39 is a membrane-anchored ecto-enzyme expressed on endothelial cells (EC), integral in maintaining the antithrombotic profile of the endothelium. This ecto-enzyme works in concert with CD73 to hydrolyze both eATP (pro-inflammatory) and ADP (pro-thrombotic) ultimately to adenosine, which is anti-inflammatory, vasodilatory, and antithrombotic. CD39 activity and adenosine signalling are disrupted in idiopathic pulmonary arterial hypertension (PAH). In this work, we explored the efficacy of endothelial cell-targeted delivery of CD39 to prevent the development of acute toxin-induced PAH in a mouse model. We generated a novel therapeutic anti-VCAM-CD39 containing an scFv recognising VCAM-1 (a receptor expressed on activated EC) fused to the soluble form of extracellular human CD39. In a mouse model of endothelial cell-toxin-induced PAH, we show that a single administration of anti-VCAM-CD39 (0.4 mg/kg IV) prevented the development of PAH-- as reflected in the preservation of right ventricular systolic pressures and the absence of right ventricular hypertrophy at day 10 when compared with controls. This protection is conferred by multiple mechanisms: IL-10-driven potentiation of heme oxygenase (HO)-1, a known inhibitor of smooth muscle proliferation; VCAM-1 blockade reduces leukocyte adhesion to the endothelium; and cytoprotective effects through adenosine signalling. Thus, anti-VCAM-CD39 is a novel bifunctional therapeutic strategy for PAH.

physiology↗

bioRxiv2025

Improvement of affinity and potency of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

As rates of antimicrobial resistance (AMR) among bacterial pathogens continue to rise, the discovery and development of novel classes of therapeutics that can serve as alternatives or adjuncts to traditional small-molecule antibiotics, such as monoclonal antibodies (mAbs), is a public health priority. Some of the most promising antigen targets for antibacterial mAbs are surface polysaccharides such as O-antigen (O-Ag), a component of the lipopolysaccharide found on the outer membrane of gram-negative bacteria. However, developing mAbs against bacterial surface polysaccharides with sufficient breadth and potency to be clinically viable is difficult in part because antibodies against polysaccharides are generally low affinity, and the challenging biochemistry of polysaccharides often precludes further affinity maturation of mAbs against these targets in vitro. Here, we use a phage display library and a whole-cell in-solution panning strategy to successfully improve the affinity of a mAb against Shigella flexneri 3a O-Ag in vitro without requiring the purification of the target antigen. We demonstrate that a single mutation can improve apparent affinity approximately 10-fold without detectably increasing polyreactivity, and increased affinity correlates with enhanced potency in antibacterial effector function and anti-virulence assays. In addition, the most potent variants also gained increased breadth, successfully coordinating complement deposition and complement-independent opsonophagocytosis against S. flexneri 3b, a serotype weakly recognized by the parent mAb. Altogether, this work represents an important first step towards expanding the antibody engineering toolkit for bacterial surface polysaccharides, which will aid the development of novel mAb therapeutics against AMR bacterial pathogens.

bioengineering↗

bioRxiv2025

Autoantibodies in patients with arrhythmogenic cardiomyopathy activate GSK-3β resulting in a loss of cardiomyocyte cohesion

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac desmosome disease, as more than 50% of affected patients carry pathogenic variants in desmosome protein-coding genes., In this study, we focused on the role and mechanisms of pathogenic and non-pathogenic autoantibodies against intercalated disc (ICD) proteins such as desmoglein2 (DSG2) in ACM patients, healthy relatives and murine ACM models. IgG fractions from ACM patients and healthy relatives, but not murine ACM model-derived or healthy control IgGs, revealed positive ICD staining. Antibodies reducing the loss of cardiomyocyte cohesion were found in three out of five ACM patients. Pathogenic autoantibodies, bound to DSG2 in hiPSC-CMs, cleaved DSG2 and reduced DSG2 interaction at the molecular level. We investigated GSK-3{beta} contribution to the loss of cardiomyocyte cohesion and observed that GSK-3{beta} reduced baseline cardiomyocyte cohesion in cultured cardiomyocytes and cardiac slices. Pathogenic ACM-IgGs activated GSK-3{beta} upstream of p38MAPK, leading to phosphorylation and junctional loss of {beta}-catenin. GSK-3{beta} inhibition rescued the loss of cell cohesion induced by ACM-IgGs in ACM hiPSC-CMs. Pathogenic autoantibodies targeting DSG2 are present in ACM patients and impair cardiomyocyte cohesion in a GSK-3{beta}-dependent manner. In contrast, autoantibodies are absent in murine ACM models and are non-pathogenic in some patients, healthy relatives.

cell biology↗

bioRxiv2025

Apremilast improves cardiomyocyte cohesion and arrhythmia in different models for arrhythmogenic cardiomyopathy

BackgroundArrhythmogenic cardiomyopathy (ACM) is a genetically inherited desmosome heart disease leading to life-threatening arrhythmias and sudden cardiac death. Currently, ACM treatment paradigms are merely symptom targeting. Recently, apremilast was shown to stabilize keratinocyte adhesion in the desmosomal disease pemphigus vulgaris. Therefore, this study investigated whether apremilast can be a therapeutic option for ACM. MethodsHuman induced pluripotent stem cells from a healthy control (hiPSC) and an ACM index patient (ACM-hiPSC) carrying a heterozygous desmoplakin (DSP) gene mutation (c.2854G>T, p.Glu952Ter), confirmed by whole exome sequencing (WES), were established. Cyclic-AMP ELISA, dissociation assay, immunostaining, and Western blotting analyses were performed in human iPSC-derived cardiomyocytes (hiPSC-CMs), murine HL-1 cardiomyocytes, and cardiac slices derived from wild-type (WT) mice, plakoglobin (PG, Jup) knockout (Jup-/-) (murine ACM model) or PG Serine 665 phosphodeficient (JUP-S665A) mice. Microelectrode array (MEA) analyses in ventricular cardiac slices and Langendorff heart perfusion were performed to analyze heart rate variability and arrhythmia. ResultsACM-hiPSC derived cardiomyocytes (ACM-hiPSC-CMs) revealed a significant loss of cohesion, which was rescued by apremilast. Further, treatment with apremilast strengthened basal cardiomyocyte cohesion in HL-1 cells and WT murine cardiac slices, paralleled by phosphorylation of PG at Serine 665 in human and murine models. In HL-1 cells, apremilast in addition activated ERK1/2, inhibition of which abolished apremilast-enhanced cardiomyocyte cohesion. Further, dissociation assays in slice cultures from JUP-S665A and Jup-/- mice revealed that PG is crucial for apremilast-enhanced cardiomyocyte cohesion. In parallel to enhanced cell adhesion, MEA and Langendorff measurements from WT and Jup-/- mice demonstrated decreased heart rate variability and arrhythmia after apremilast treatment. ConclusionsApremilast improves loss of cardiomyocyte cohesion, enhances localization of DSG2, and reduces arrhythmia in human and murine models of ACM ex vivo and in vitro, providing a novel treatment strategy for ACM by preserving desmosome function. Translational perspectiveThe current therapeutic options for patients with arrhythmogenic cardiomyopathy (ACM) include lifestyle changes, treatment with anti-arrhythmic drugs, catheter ablation, implantable cardiac defibrillators, and ultimately, heart transplantation for patients who are having therapy refractory arrhythmia or developed heart failure. However, lifestyle changes, such as restraining from physical endurance activities and {beta}-blocker therapy, are most used in patients carrying genetic variants coding for proteins of the desmosomal complex. Recent advancements hint that strategies enhancing intracellular cAMP could be beneficial in treating desmosomal diseases and can be effective therapeutics, which would be highly relevant for ACM patients. In this study, we show that apremilast improves loss of cardiomyocyte cohesion, enhances localization of desmosomal proteins, and reduces arrhythmia in both human and murine models of ACM ex vivo and in vitro, providing a novel treatment strategy for ACM by preserving desmosome function.

cell biology↗

bioRxiv2025

Characterization of transcriptomic changes across Coccidioides morphologies using RiboMarker(R)-enhanced RNA sequencing

Coccidioides is a dimorphic, pathogenic fungus responsible for transmission of the mammalian disease colloquially known as "Valley fever". To better understand the molecular basis of Coccidioides pathogenesis, previous studies have characterized transcriptomes that define transitions between the saprobic and pathogenic life stages of the two species that cause Valley fever - Coccidioides immitis and Coccidioides posadasii. However, none of these studies have focused on small RNA profiles, which have been shown in several pathogenic fungi to play crucial roles in host-pathogen communication, affecting virulence and infectivity. In this study, we analyzed changes in small RNA expression across three major morphologies of C. posadasii: arthroconidia, mycelia, and spherules, from both intracellular and extracellular fractions. Utilizing RiboMarker(R) small RNA and RNA fragment library preparation, we show enhanced coverage across the transcriptome by increasing incorporation of normally incompatible RNAs into the sequencing pool. Using these data, we observed transcriptomic shifts during the transition of arthroconidia to either mycelia or spherules, marked largely by changes in both protein-coding, tRNA, and unannotated loci. As little is known regarding the mechanisms governing these life stage transitions, these data provide better insight into those small RNA- and fragment-producing genes and loci that may be required for progression between Coccidioides saprobic and parasitic life cycles. Additionally, analysis of fragmentation patterns across all morphologies suggests unique patterns of RNA fragmentation across a cohort of RNA species that correlate with a given ecotype. Finally, we noted evidence of RNA export to the extracellular space, particularly regarding snRNA and tRNA-derived fragments as well as mRNA-derived transcripts, during the transition to either mycelia or spherules, which may play roles in cell-cell, and/or host-pathogen communication. Going forward, this newly established intra- and extracellular Coccidioides sRNA atlas will provide a foundation for potential biomarker discovery and contribute to our understanding of the molecular basis for virulence in Valley fever.

molecular biology↗

bioRxiv2024

A direct interaction between the RNA-binding proteins Staufen and Tm1-I/C regulates oskar mRNP composition and transport

In the Drosophila female germline, oskar messenger RNA is transported on microtubules from the nurse cells to the posterior pole of the oocyte, where it is translated. Transport of oskar transcripts from the nurse cells into the oocyte requires dynein, while localization of the mRNAs within the oocyte to the posterior pole is dependent upon kinesin-1. Staufen, a dsRNA-binding protein, has been shown to bind the oskar mRNA transport complex in the oocyte and inactivate dynein; however, it remains unclear how kinesin is activated. Here, using surface plasmon resonance, nuclear magnetic resonance spectroscopy and RNA imaging within egg chambers, we demonstrate that Staufen directly interacts with Tm1, a non-canonical kinesin adaptor. This work provides a molecular explanation for the previously unclear role of Staufen in oskar mRNA localization.

molecular biology↗

bioRxiv2024

Evolutionary divergence of drug-binding sites in bacterial ribosomes

Ribosomes from certain bacteria possess divergent drug-binding sites compared to those of Escherichia coli, leading to natural evasion or hypersensitivity to antibiotics. However, in the absence of systematic studies, it is unknown whether this observed divergence is a rare exception or a common occurrence among bacterial species. Here, we address this question by reconstructing the evolutionary history of drug-binding residues of the ribosome from the origin of bacteria to the present day. This analysis reveals the extent of natural diversity of ribosomal drug-binding sites between bacterial species, which may inform the development of species-specific antimicrobials and a more accurate and personalized choice of ribosome-targeting drugs for a given pathogen.

microbiology↗

bioRxiv2024

ARTIC RSV amplicon sequencing reveals global RSV genotype dynamics.

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections (LTRIs) in young children and adults over 65, contributing significantly to global healthcare burdens. With the recent approval of multiple pharmacological interventions for RSV, there is an increased demand for efficient, high-throughput sequencing methods to monitor RSV genetic diversity and any potential impact these interventions may have. Here we introduce two novel amplicon-based sequencing schemes designed for RSV A and B, optimised for integration with widespread existing ARTIC sequencing workflows. We demonstrate that these primer schemes can produce high quality genomes from RSV samples across the globe, with eight laboratories in five countries generating complete genomes on both Nanopore and Illumina sequencing platforms. The ability to effectively multiplex these RSV A and B primer schemes, enables streamlined, high-throughput sequencing without prior subtyping. Furthermore, these results provide a snapshot of the circulating diversity of RSV. Phylogenetic analysis of the 882 samples sequenced for this study suggests only minimal geographic clustering of RSV sequences, underscoring the global nature of RSV spread. It also highlights the distinct lineage dynamics seen between RSV A and B. This study represents an advancement in RSV genomics, providing robust tools for global sequencing efforts aimed at tracking RSV evolution and assessing the efficacy of new therapeutic interventions both rapidly and at scale.

genomics↗

bioRxiv2024

High-content high-resolution microscopy and deep learning assisted analysis reveals host and bacterial heterogeneity during Shigella infection

Shigella flexneri is a Gram-negative bacterial pathogen and causative agent of bacillary dysentery. S. flexneri is closely related to Escherichia coli but harbors a virulence plasmid that encodes a Type III Secretion System (T3SS) required for host cell invasion. Widely recognized as a paradigm for research in cellular microbiology, S. flexneri has emerged as important to study mechanisms of cell-autonomous immunity, including septin cage entrapment. Here we use high-content high-resolution microscopy to monitor the dynamic and heterogeneous S. flexneri infection process by assessing multiple host and bacterial parameters (DNA replication, protein translation, T3SS activity). In the case of infected host cells, we report a reduction in DNA and protein synthesis together with morphological changes that suggest S. flexneri can induce cell-cycle arrest. We developed an artificial intelligence image analysis approach using Convolutional Neural Networks to reliably quantify, in an automated and unbiased manner, the recruitment of SEPT7 to intracellular bacteria. We discover that heterogeneous SEPT7 assemblies are recruited to bacteria with increased T3SS activation. Our automated microscopy workflow is useful to illuminate diverse host and bacterial interactions at the single-cell and population level, and to fully characterise the intracellular microenvironment controlling the S. flexneri infection process.

cell biology↗