Biology subjects

Patel, B.

Publications and source records attributed to Patel, B..

At least 19 recordsLinked to original sources

bioRxiv2026

SpiE is a distinct gut microbial 4-cholesten-3-one 5β-reductase that acts with SpiR to convert cholesterol to coprostanol

Gut microbes convert cholesterol into coprostanol, a poorly absorbed sterol that is largely excreted in feces and is linked to lower serum cholesterol levels. However, the enzymes responsible for this process remain poorly characterized. The second step of the pathway, cholestenone reduction into coprostanone, has long been established, yet the enzyme(s) responsible were unknown. Here, we use comparative genomics to identify SpiE, a cholestenone (4-cholesten-3-one) reductase that is part of the steroid processing (spi) pathway with SpiR. We confirm that SpiE binds to and reduces both cholestenone and progesterone into 5{beta}-reduced forms. SpiR and SpiE together convert cholesterol into coprostanol, indicating that both enzymes are sufficient for coprostanol formation. SpiE belongs to the short-chain dehydrogenase (SDR) family and represents an evolutionarily distinct lineage of SDR ene-reductases. Metagenomic analyses reveal that spiE relative abundance is strongly correlated with stool coprostanol (Spearman's {rho}=0.81, p=5.99e-65). In longitudinal patient cohorts, antibiotic treatment coincided with loss of SpiE and coprostanol production. Post antibiotics, the emergence of distinct SpiE-encoding genera rescued coprostanol production in some individuals. Our findings provide the biochemical basis of the cholesterol to coprostanol pathway and lay the groundwork for future studies linking it to host physiology.

microbiology↗

bioRxiv2026

Individual mouse mitotic chromosomes exhibit cell type-specific differences in biomechanical properties

Cyclic episodes of chromosome compaction and de-condensation are features of eukaryotic cell division that aid mitotic segregation and help prevent aneuploidy. While biophysical data on mitotic chromosome structure has been previously obtained, heterogeneity within samples can confound analyses and precludes direct like-for-like comparisons. To circumvent this, we employed advanced flow cytometry to purify specific metaphase chromosomes with biotinylated telomeres from stably engineered mouse cells. We show that ESC-derived metaphase chromosomes 3 and 19 display distinct properties but share a conserved force-dependent mechanical response. In contrast, chromosome equivalents isolated from NSCs and preB cells show markedly different force-dependent responses, reflecting progressive differentiation stages. Covalent crosslinking of ESC-derived chromosomes alters biomechanical properties to mimic equivalents from more differentiated cells. Collectively, these results highlight the need to isolate specific, homogeneous metaphase chromosome samples to accurately decipher their complex behaviours.

biophysics↗

bioRxiv2026

ASS1 deficiency defines a therapeutic vulnerability in Philadelphia chromosome-positive acute lymphoblastic leukaemia

Amino acid deprivation with L-asparaginase is a cornerstone of treatment in acute lymphoblastic leukaemia (ALL), but clinical challenges limit its use in adults. Deficiency in the enzyme argininosuccinate synthase (ASS1-low) confers arginine auxotrophy, defining a dependence on extracellular arginine, and represents an analogue metabolic vulnerability that is targetable through arginine deprivation. We analysed transcriptomic data across >550 adult B-ALL cases to establish clinico-biologic characteristics of low ASS1 expression identifying the Philadelphia chromosome-positive (Ph+) ALL subgroup as a stereotypical arginine auxotroph within molecularly diverse B-ALL. Functionally, arginine deprivation with pegargiminase induced robust apoptosis in both Ph+ ALL cell lines and primary samples in an ASS1-dependant manner and was highly effective as a monotherapy treatment in independent Ph+ ALL patient derived xenografts. Mechanistically, arginine deprivation induces endoplasmic reticulum stress mediated apoptosis which was orthogonal to tyrosine kinase inhibition (TKI) mediated outcomes. Using in vitro and in vivo models of non-genetically mediated TKI-resistance, we demonstrate pegargiminase and TKI combinations robustly eradicates TKI-resistant leukaemia. Thus, we establish ASS1 deficiency (ASS1-low) as a therapeutically actionable vulnerability in Ph+ ALL and a strategy to bypass TKI-resistance, supporting clinical evaluation of arginine deprivation as a novel adjunct to chemotherapy-free treatment.

cancer biology↗

bioRxiv2026

Inhibition of miR-1307 Reverses Resistance to Cisplatin in Drug-Resistant Oral Squamous Cell Carcinoma

BackgroundChemo-resistance remains a major clinical challenge in Oral Squamous Cell Carcinoma (OSCC), attributed to the intrinsically resistant cells. Although tumour-derived extracellular vesicles (EVs) have been implicated in cell-cell communication, their role in propagating chemo-resistance remains poorly defined. This study aims to identify salivary EV-associated miRNAs capable of predicting chemoresistance and to delineate the role of miR-1307-5p in modulating CSC-driven therapeutic refractoriness. MethodsSalivary EV-derived expression profile of miR-1307-5p was assessed by qPCR in chemo resistant OSCC patients and further validated in TCGA small RNA sequencing datasets. Expression was validated by qPCR and correlated with clinicopathological outcomes. Functional assays including cell-cycle analysis, apoptosis, migration/invasion, 3D spheroids, angiogenesis, and CAM assays were performed in miR-1307-5p inhibited CD44 CSC subpopulation compared to its vehicular control. Transcriptomic profiling cross-referencing with TCGA was conducted to identify potential novel targets of miR-1307-5p. Chemo-sensitisation was assessed by treating the knockdown chemo resistant cells with low dose cisplatin and validating it using in-vitro functional assays and orthotopic xenograft model. ResultsmiR-1307-5p was significantly elevated in salivary EVs of chemo resistant OSCC patients and correlated with poor overall survival (p = 0.03). The miRNA was markedly enriched in endogenously resistant CD44 CSCs. Silencing of miR-1307-5p induced G2/M arrest, triggered apoptosis, impaired invasion, and reduced angiogenesis both in-vitro and in ex-vivo assays. Transcriptomic profiling, TCGA validation, and integrative pathway analysis identified key oncogenic hubs which converge on PI3K-AKT, MAPK/ERK, and YAP signalling pathways governing EMT. Inhibition of miR-1307-5p restored cisplatin sensitivity in resistant CSCs, with low-dose cisplatin producing substantial tumour suppression in-vitro and in-vivo. Reduced CD44 expression in xenograft models confirmed CSC reprogramming. EVs from anti-miR-treated cells confer chemo sensitisation upon uptake by resistant CSCs. Xenograft models substantiated that EVs can initiate tumour formation and that EV-mediated delivery of anti-miR-1307-5p drives significant tumour regression. ConclusionThis study identifies salivary EV-derived miR-1307-5p as a clinically relevant biomarker of chemoresistance in OSCC and reveals its mechanistic role in sustaining CSC-driven therapeutic failure. Targeting miR-1307-5p offers a promising avenue for restoring cisplatin sensitivity and developing exosome-based therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/709730v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@19f88e0org.highwire.dtl.DTLVardef@d36b95org.highwire.dtl.DTLVardef@3c2579org.highwire.dtl.DTLVardef@c04ef5_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Chemoproteomics discovery of a CNS-penetrant covalent inhibitor of PIKfyve

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a CNS-penetrant covalent PIKfyve inhibitor, DUN058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centered approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, the development of DUN058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.

biochemistry↗

bioRxiv2026

Antigen presentation requirements for effective cDC1-based cancer immunotherapy

Type 1 conventional dendritic cells (cDC1s) are important for generating and sustaining antitumor immunity. Accordingly, the abundance of cDC1s in human tumors correlates with improved outcomes in cancer. Capitalizing on this role, we previously demonstrated that vaccination with in vitro-derived murine cDC1s elicits durable tumor control in multiple preclinical models; however, the immunological mechanisms underlying the efficacy of cDC1 vaccination remain unclear. Here, we examined whether in vitro-derived cDC1s resemble tumor-infiltrating DC populations and whether MHC-I and MHC-II antigen presentation contribute to cDC1-mediated tumor control following vaccination in melanoma. As expected, MHC-I- or MHC-II-deficiency had minimal impact on the transcriptional state of cDC1s in homeostasis or following stimulation with the adjuvant poly dI:dC. Moreover, in vitro-derived cDC1s cultured under steady-state conditions closely resembled tumor-infiltrating cDC1s, whereas their poly dI:dC-stimulated counterparts resembled CCR7+ tumor-infiltrating DC populations, also referred to as mregDCs or LAMP3+ DCs. Our data further show that both MHC-I and MHC-II contribute to tumor control upon cDC1 vaccination, and coexpression of MHC-I and MHC-II on the same cDC1 is necessary for a robust vaccine response. We also identified an important function for host cDC1s in supporting the efficacy of vaccination with in vitro-derived cDC1s, as judged by impaired tumor control in Irf8+32-/- mice, which lack endogenous cDC1s. Overall, these results indicate that effective antitumor responses depend on MHC-I and MHC-II antigen presentation by vaccine-delivered cDC1s, with additional contributions from host cDC1s. Key pointsO_LIIn vitro-generated cDC1s resemble intratumoral DC populations found in mice and humans. C_LIO_LIMHC-I and MHC-II antigen presentation by vaccine-delivered cDC1s contribute to antitumor efficacy. C_LIO_LICoexpression of MHC-I and MHC-II on the same cDC1 enhances vaccine responses. C_LIO_LIAntitumor responses reflect the activity of vaccine and endogenous cDC1s. C_LI

immunology↗

bioRxiv2025

Dataset Documentation for Responsible AI: Analysis of Suitability and Usage for Health Datasets

Artificial Intelligence (AI) is rapidly transforming healthcare, but also raising concerns about algorithmic biases that mostly stem from the training data. It is widely supported that transparent dataset documentation is key to enabling responsible AI development. Several standardized dataset documentation approaches have been established, such as Datasheet, Dataset Nutrition Label, Accountability Documentation, Healthsheet, and Data Card. However, their suitability and usage for health datasets remain unclear. In this work, we compared all five approaches and evaluated their alignment with the STANDING Together Recommendations for Documentation of Health Datasets. We also investigated their real-world usage and gathered insights from generators and consumers of health datasets. Our findings reveal that none of these documentation approaches are used widely or fully suited for health datasets. We recommend developing a standard documentation approach for health datasets along with clear guidelines and automation tools to support adoption.

bioinformatics↗

bioRxiv2025

Lymphotoxin-driven cancer cell eradication by tumoricidal CD8+ tumor-infiltrating lymphocytes

Tumor-infiltrating lymphocyte (TIL) therapy is FDA-approved for patients with treatment-resistant advanced melanoma, but the TIL subpopulations critical for tumor eradication remains incompletely understood. Using patient-derived TIL-melanoma co-cultures, we identified and characterized a novel subset of CD8+ TIL, capable of class I HLA-independent cancer cell lysis. The lymphotoxin {beta} receptor (LT{beta}R) and interferon (IFN) sensing pathways were nominated as key determinants of TIL-mediated cancer cell killing from a whole-genome, loss-of-function CRISPR screen. Validation studies confirmed that dual LT{beta}R and IFN sensing is necessary and sufficient for cancer cell lysis, and that expanded CD8+ TIL express high lymphotoxin {beta} (LTB) and upregulate lymphotoxin (LTA) upon coculture with cancer cells. Leveraging paired scRNA-seq and scTCR-seq data, we confirmed that enrichment of LTB+CD8+ T cells is associated with clinical response to TIL, and that LTB+CD8+ TIL are expanded from putative neoantigen-reactive, LTBlo CD8+ T cells in resected tumors. SignificanceWe have uncovered a previously unrecognized mechanism of TIL-mediated tumor eradication, providing mechanistic insights into the role of LTBR/IFN signaling in TIL-mediated cancer cell killing, and potentially offering insights into novel strategies to isolate, enrich, and expand tumoricidal TIL or augment specific TIL functions to enhance tumor control.

cancer biology↗

bioRxiv2025

Nanoscopic tau aggregates in Parkinson's disease

Post-mortem tau pathology is frequently observed in Parkinsons disease (PD) using immunohistochemistry (IHC) to measure large inclusions, however, small protein aggregates that precede inclusions are considered a major driver of toxicity in neurodegenerative disease. We aimed to uncover the distribution of nanoscopic aggregates across six brain regions in post-mortem tissue from 14 PD and 15 controls using the single-molecule pull-down assay (SiMPull). In the hippocampus and amygdala, tau IHC and SiMPull were associated with advanced age in controls and dementia status in PD. Despite negligible tau IHC-labelled aggregates in the putamen, we identified a unique population of high-intensity nanoscopic tau aggregates for a subset of PD cases using SiMPull, ranging from 10-1,000 epitopes per aggregate and 30-1,000 nm in length. Previous evidence linking nigrostriatal tau pathology and motor deficits indicates that the nanoscopic tau aggregates identified in this study may contribute to striatal dysfunction in PD.

neuroscience↗

bioRxiv2025

Phase Separation Oppositely Modulates G-quadruplex and i-Motif DNA Folding in the Nuclei of Living Cells

G-quadruplexes and i-motifs are non-canonical DNA structures that are important not only as targets of gene regulation in cancer therapy, but also as nanotools with biomedical applications. However, their apparent in vitro stabilities and dynamics cast doubt upon their physiological relevance and utility in vivo. Here we investigate folding of the thrombin-binding aptamer G-quadruplex and the TAA(C5TAA)4 i-motif. We use fast relaxation imaging, which couples fluorescence microscopy with laser-induced temperature jumps, to quantify the stability and dynamics of FRET-labelled constructs in vitro and in the nuclei of living cells. Our work suggests that the stability and kinetics of i-motif and G-quadruplex DNA are carefully tuned by phase separation to fit the seconds-to-minutes timescales of key regulatory processes inside cells. Taken together, our findings underscore the importance of studying the folding of DNA structures under near-physiological conditions to their mechanistic understanding as well as for the development of DNA-targeted drugs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/661982v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@e76eedorg.highwire.dtl.DTLVardef@23aefforg.highwire.dtl.DTLVardef@b6117aorg.highwire.dtl.DTLVardef@135736a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

bioRxiv2025

Mapping the spatial architecture of glioblastoma from core to edge delineates niche-specific tumor cell states and intercellular interactions

Treatment resistance in glioblastoma (GBM) is largely driven by the extensive multi-level heterogeneity that typifies this disease. Despite significant progress toward elucidating GBMs genomic and transcriptional heterogeneity, a critical knowledge gap remains in defining this heterogeneity at the spatial level. To address this, we employed spatial transcriptomics to map the architecture of the GBM ecosystem. This revealed tumor cell states that are jointly defined by gene expression and spatial localization, and multicellular niches whose composition varies along the tumor core-edge axis. Ligand-receptor interaction analysis uncovered a complex network of intercellular communication, including niche- and region-specific interactions. Finally, we found that CD8 positive GZMK positive T cells colocalize with LYVE1 positive CD163 positive myeloid cells in vascular regions, suggesting a potential mechanism for immune evasion. These findings provide novel insights into the GBM tumor microenvironment, highlighting previously unrecognized patterns of spatial organization and intercellular interactions, and novel therapeutic avenues to disrupt tumor-promoting interactions and overcome immune resistance.

cancer biology↗

bioRxiv2025

Physiological analysis of the mechanism of Ci transcription factor activation through multiple Fused phosphorylation sites in Hedgehog signal transduction

Hedgehog (Hh) proteins elicit dose-dependent transcriptional responses by binding Patched receptors to activate transmembrane Smoothened (Smo) proteins. Activated Smo inhibits Ci/Gli transcription factor phosphorylation by Protein Kinase A (PKA) and consequent proteolytic processing to repressor forms; it also promotes nuclear transport and activity of full-length Ci/Gli proteins to induce Hh target genes. Smo-activated Fused (Fu) kinase drives Ci activation in Drosophila, while Suppressor of Fused (Su(fu)) counters full-length Ci/Gli activity and stabilizes full-length Ci/Gli by direct binding to at least three surfaces. Here, we used CRISPR-generated designer ci alleles to investigate alterations to Fu phosphorylation sites and to regions around Ci-Su(fu) interfaces under physiological conditions in Drosophila imaginal wing discs. Surprisingly, we identified alterations that activate Ci without significant loss of stabilization by Su(fu) and contributions of multiple Fu target sites to Ci activation in the absence of Su(fu), suggesting that the affected sites mediate Ci activation by regulating Ci-Ci, rather than Ci-Su(fu) interactions. We propose that those interactions maintain full-length Ci in a closed conformation that also facilitates, and is stabilized by, cooperative Ci-Su(fu) binding. Access to binding partners necessary for Ci activation is promoted through phosphorylation of at least four Fu sites on Ci, likely by directly disrupting Ci-Ci contacts and one Ci-Su(fu) interface without substantial Ci-Su(fu) dissociation, contrary to previous proposals. We also found that the Ci binding partner, Costal 2 (Cos2), which silences Ci in the absence of Hh, can facilitate Ci activation by Fu kinase.

developmental biology↗

bioRxiv2025

Fos expression in the periaqueductal gray, but not the ventromedial hypothalamus, is correlated with psychosocial stress-induced cocaine-seeking behavior in rats

Psychosocial stressors are known to promote cocaine craving and relapse in humans but are infrequently employed in preclinical relapse models. Consequently, the underlying neural circuitry by which these stressors drive cocaine seeking has not been thoroughly explored. Using Fos expression analyses, we sought to examine whether the ventromedial hypothalamus (VMH) or periaqueductal gray (PAG), two critical components of the brains hypothalamic defense system, are activated during psychosocial stress-induced cocaine seeking. Adult male and female rats self-administered cocaine (0.5 mg/kg/inf IV, fixed-ratio 1 schedule, 2 h/session) over 20 sessions. On sessions 11, 14, 17, and 20, a tactile cue was present in the operant chamber that signaled impending social defeat stress (n=16, 8/sex), footshock stress (n=12, 6/sex), or a no-stress control condition (n=12, 6/sex) immediately after the sessions conclusion. Responding was subsequently extinguished, and rats were tested for reinstatement of cocaine seeking during re-exposure to the tactile cue that signaled their impending stress/no-stress post-session event. All experimental groups displayed significant reinstatement of cocaine seeking, but Fos analyses indicated that neural activity within the rostrolateral PAG (rPAGl) was selectively correlated with cocaine-seeking magnitude in the socially-defeated rats. rPAGl activation was also associated with active-defense coping behaviors during social defeat encounters and with Fos expression in prelimbic prefrontal cortex and orexin-negative cells of the lateral hypothalamus/perifornical area in males, but not females. These findings suggest a potentially novel role for the rPAGl in psychosocial stress-induced cocaine seeking, perhaps in a sex-dependent manner.

neuroscience↗

bioRxiv2024

Characterizing Behavioral Effects of Early-Life Stress in an Animal Model of Auditory Processing

Animal models provide significant insight into the development of typical and disordered sensory processing. Such models have been established to take advantage of physical and behavioral characteristics of specific species. For example, the Mongolian gerbil is a well-established model for auditory processing, with a hearing range similar in frequency to that of humans and an easily accessible cochlea. Recently, early-life stress (ELS) has been shown to affect sensory processing in auditory, visual, and somatosensory neural regions. To understand the functional impact of ELS, it is necessary to evaluate the susceptibility of sensory perceptual abilities to this early perturbation. Yet measuring sensory perception - e.g., using operant conditioning - often concurrently involves animal behavioral elements such as attention, memory, learning, and emotion. All of these elements are well-known to be impacted by ELS, and may affect behavioral measurements in ways that could be misconstrued as sensory deficits. Thus, it is critical to characterize which behavioral elements are affected by ELS in any sensory model. Here we induced ELS during a developmental time window for maturation of the auditory cortex in Mongolian gerbils. We conducted behavioral measures in juveniles, a developmental age when ELS is known to impair the auditory pathway. ELS had no effect on overall activity but reduced anxiety-related behavior, impaired recognition memory, and improved spatial memory, with some sex-specific effects. These effects may influence the ability of gerbils to learn and retain operant training, particularly if anxiety-provoking reinforcement is used.

animal behavior and cognition↗

bioRxiv2024

AI-readiness for Biomedical Data: Bridge2AI Recommendations

Biomedical research is rapidly adopting artificial intelligence (AI). Yet the inherent complexity of biomedical data preparation requires implementing actionable, robust criteria for ethical and explainable AI (XAI) at the "pre-model" stage, encompassing data acquisition, detailed transformations, and ethical governance. Simple conformance to FAIR (Findable, Accessible, Interoperable, Reusable) Principles is insufficient. Here, we define criteria and practices for reliable AI-readiness of biomedical data, developed by the NIH Bridge to Artificial Intelligence (Bridge2AI) Standards Working Group across seven core dimensions of dataset AI-readiness: FAIRness, Provenance, Characterization, Ethics, Pre-model Explainability, Sustainability, and Computability. Conformance to these criteria provides a basis for pre-model scientific rigor and ethical integrity, mitigating downstream risks of bias and error prior to AI modeling. We apply and evaluate these standards across all four Bridge2AI flagship datasets, spanning functional genomics to clinical medicine, and encode them in machine-actionable metadata bound to the datasets. This framework sets a benchmark for preparing ethical, reusable datasets in biomedical AI and provides standardized methods for reliable pre-model data evaluation.

bioinformatics↗

bioRxiv2024

Plasmid conjugation drives within-patient plasmid diversity

Plasmids are well known vehicles of antimicrobial resistance (AMR) genes dissemination. Through conjugation, plasmid-encoded AMR genes are spread among neighbouring bacteria, irrespective of their strain or even their species. This process is very concerning from a public health perspective, as plasmid-borne AMR gene outbreaks are often not confined to single species or strains and are therefore more difficult to be fully uncovered. At the moment, the impact of plasmid conjugation on within-patient plasmid diversity is not well understood. In this work we will tackle the role of conjugation on within-patient plasmid diversity using a dataset of carbapenemase-producing Enterobacterales (CPEs). The dataset of 256 sequences from 115 patients was sampled across England over 30 months. Each patient has more than one sequence, with at least one sequence carrying an OXA-48 gene, a well-known plasmid-borne carbapenemase-encoding gene. If more than one sequence carries the OXA-48 gene, they are carried on different bacterial hosts. Using a hybrid de novo-on-reference assembly pipeline, we were able to reconstruct the full OXA-48 plasmid from short read sequencing data for 232 of the 256 sequences. Of the 115 patients, 83 (72%) of patients had an identical OXA-48 plasmid in two or more sequences. Only 2 patients carried very different (>200 SNPs) alleles of the OXA-48 plasmid, probably from separate acquisitions. Our study shows that when more than one bacterial host carrying an OXA-48 plasmid is found in a patient, it is most likely that the same plasmid has been shared via conjugation. The event of separate acquisition of different plasmids in different bacterial hosts is highly unlikely in our dataset. Data StatementWe use data provided by Hopkins et al 2022 [16]. The data can be accessed from the National Center for Biotechnology Information (NCBI) and can be found at Bioproject Accession no. PRJNA788733. None of the data used was synthetically generated. Impact StatementConjugative plasmids are well known vessels of horizontal gene transfer, with a prominent role in the spread of antimicrobial resistance genes among different bacterial species or strains. At the epidemiological level, conjugation combined with sequencing a single colony per patient, results in plasmids outbreaks carrying antimicrobial resistance genes being found in different bacterial species and strains in different patients, potentially eluding surveillance protocols based on same bacterium/same resistance scheme. In this study we analyse within-patient plasmid diversity in a dataset with more than one sequence per patient. Only two patients show clear genomic signs of separate plasmids acquisition, while 83 patients share identical plasmids in different bacterial hosts. This points out to a very strong role of plasmid conjugation in shaping within-patient plasmid diversity.

bioinformatics↗

bioRxiv2024

Extracellular vesicles regulate metastable phenotypes of lymphangioleiomyomatosis cells via shuttling ATP synthesis to pseudopodia and activation of integrin adhesion complexes.

Pulmonary lymphangioleiomyomatosis (LAM) is metastatic sarcoma but mechanisms regulating LAM metastasis are unknown. Extracellular vesicle (EV) regulate cancer metastasis but their roles in LAM have not yet been investigated. Here, we report that EV biogenesis is increased in LAM and LAM EV cargo is enriched with lung tropic integrins, metalloproteinases, and cancer stem cell markers. LAM-EV increase LAM cell migration and invasion via the ITG6/{beta}1-c-Src-FAK-AKT axis. Metastable (hybrid) phenotypes of LAM metastasizing cells, pivotal for metastasis, are regulated by EV from primary tumor or metastasizing LAM cells via shuttling ATP synthesis to cell pseudopodia or activation of integrin adhesion complex, respectively. In mouse models of LAM, LAM-EV increase lung metastatic burden through mechanisms involving lung extracellular matrix remodeling. Collectively, these data provide evidence for the role of EV in promoting LAM lung metastasis and identify novel EV-dependent mechanisms regulating metastable phenotypes of tumor cells. Clinical impact of research is that it establishes LAM pathway as novel target for LAM therapy.

cancer biology↗