Biology subjects

Roy, A.

Publications and source records attributed to Roy, A..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Targeting Metabolic Vulnerabilities: Valinomycin Augments the Potency of Bioenergetic Inhibitors in Combatting Drug-Resistant and Dormant Mycobacterium tuberculosis

Tuberculosis (TB) treatment is hampered by monotherapy limitations and phenotypic drug tolerance, citing the need for effective drug combinations. The mycobacterial electron transport chain (ETC), crucial for oxidative phosphorylation and ATP production in dormant Mycobacterium tuberculosis (Mtb), is a key target. This study investigates combining established bioenergetic inhibitors-bedaquiline (BDQ), telacebec (Q203), and clofazimine (CFZ) with the potassium ionophore valinomycin, which disrupts the proton motive force (pmf). We demonstrated that valinomycin significantly potentiated the anti-TB activity of these inhibitors against both replicating and nutrient-starved non-replicating Mtb. Checkerboard assays revealed synergistic activity with BDQ and additive effects with Q203 and CFZ, correlated with a two to three-fold reduction in ATP IC50 values. Critically, valinomycin converted the bacteriostatic activity of the inhibitors at sub-MIC concentration into bactericidal killing in time-dependent killing assay, achieving sterilization. This lethal synergy for the combinations was also observed in a THP-1 macrophage intracellular model for TB. Respiration assays confirmed that the combinations collectively halted oxygen consumption. We conclude that concurrently targeting specific ETC on Mtbs bioenergetics. This strategy, particularly the BDQ/valinomycin synergy, represents a promising cornerstone for developing novel sterilizing regimens to shorten TB therapy and overcoming drug tolerance.

microbiology↗

bioRxiv2025

Substitution of a valine to glutamic acid in the omega-like loop of MSMEG_6194 of Mycobacterium smegmatis interchanges its activity from DD-carboxypeptidase to beta-lactamase

The genome of Mycobacterium smegmatis encodes numerous penicillin-interacting enzymes, and analysing their functions provides insights into the evolutionary mechanisms behind beta-lactam resistance in mycobacteria. In this study, we characterised one such enzyme, MSMEG_6194, annotated as a putative beta-lactamase. Although MSMEG_6194 shares structural similarity with class A beta-lactamases, it showed no detectable beta-lactamase activity under the tested conditions. Heterologous expression of MSMEG_6194 in Escherichia coli and{Delta} msmeg_6194 deleted strains of M. smegmatis did not confer significant resistance to beta-lactams, and the purified protein failed to hydrolyse nitrocefin either. However, ectopic expression of MSMEG_6194 partly restores the morphological defects in seven PBP-deleted E. coli strains, and the purified enzyme successfully cleaves the terminal D-alanine from a pentapeptide substrate, confirming its DD-carboxypeptidase activity. Structural analysis revealed the absence of a conserved glutamic acid residue in the omega-loop, which is critical for beta-lactamase catalysis in class A beta-lactamase. Substituting this residue (V139E mutant) imparts beta-lactamase activity though significantly reduces DD-carboxypeptidase function. Overall, these findings establish MSMEG_6194 as a DD-carboxypeptidase and demonstrate how a single amino acid change can alter catalytic preference, shedding light on the evolutionary transition from DD-Carboxypeptidases to beta-lactamases in mycobacteria.

molecular biology↗

bioRxiv2025

Apoptosis reveals human evolutionary heritage sequestered in non-coding DNA

Cell-free chromatin particles (cfChPs), primarily derived from non-coding DNA (ncDNA) and released from apoptotic cells into the bloodstream, can be horizontally transferred into living cells. However, their potential role in generating ncDNA within recipient cells remains unknown. We previously reported that cfChP- sized fragments generated upon sonicating high-molecular-weight DNA can indiscriminately transmit themselves into foreign cells across species and kingdom boundaries. We hypothesized that cfChP-like DNA-protein complexes generated following organismal death have been exchanged among species and have progressively accumulated to form a dense patchwork that now constitutes the ncDNA, and that apoptosis may dissociate these complexes, rendering them amenable to detection. To test this hypothesis, we used species-specific DNA fluorescent in situ hybridisation probes and antibodies against archaea, eubacteria, plants, algae, fungi, protozoa, Drosophila, fish, chicken, mouse, rat, pig, dog, and monkey on intact and apoptotic human cells. We detected no reactivity with intact cells but strong reactivity with the ncDNA component of apoptotic cells. These findings indicate that ncDNA represents a dense conglomeration of DNA-protein complexes derived from different species that become detectable following apoptosis. This suggests that human evolutionary heritage is conserved within ncDNA formed through progressive accumulation of genetic fragments transferred horizontally following organismal death.

evolutionary biology↗

bioRxiv2025

A switch from histone methyltransferase-EZH2 to demethylase KDM6A activity marks reinitiation of proliferation of drug treated colorectal cancer cells

Colorectal cancer (CRC) is one of the deadliest cancers, ranking third in cancer incidence worldwide. These tumor cells often adopt unique strategies under drug stress to attain a reversible drug-tolerant state and evade cell death. However, the molecular adaptations associated with this transitory emergence of the drug-tolerant state remain elusive. Herein, epigenetic alterations often dictate such reversible dynamic changes, and this study aims to characterize the role of specific epigenetic modifiers governing CRC cell survival under drug pressure and their subsequent relapse. We observed that under drug stress there is a drastic increase in the histone-repressive mark-H3K27me3, linked to an enhanced expression of EZH2 driving transcriptional inhibition of cell proliferation-associated genes and a proliferative arrest. Interestingly, drug-induced oxidative stress increased the expression of the P65 protein, which was found to interact and regulate EZH2 expression. Quenching of ROS, drug vacation, or P65 inactivation compromised EZH2 activity concurrent with a re-initiation of cell proliferation. Interestingly, this reversal to proliferative state was associated with an elevated activity of the histone lysine demethylase-KDM6A. The promoter elements of the proliferative genes were now occupied by KDM6A instead of EZH2. Accordingly, a genetic knockdown or pharmacological inhibition of KDM6A in vitro not only resulted in increased cell death but also prevented emergence of the re-proliferative CRC cells. Furthermore, KDM6A inhibition in combination with chemotherapy drug, resulted in an increased tumor regression in vivo. Our study thus highlights the importance of KDM6A as a therapeutic target in preventing CRC growth and relapse which can have future therapeutic implications.

cancer biology↗

bioRxiv2025

TAK1 is a key regulator of oncogenic signaling and differentiation blockade in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a malignant soft tissue sarcoma with a skeletal muscle phenotype, accounting for approximately 50% of all pediatric soft tissue sarcomas and 8% of all childhood cancers. Although RMS cells express myogenic regulatory factors, they fail to undergo terminal differentiation into mature muscle cells. Transforming growth factor {beta}-activated kinase 1 (TAK1) is a major signaling protein that activates multiple intracellular pathways in response to growth factors, cytokines, and microbial products. Emerging evidence suggests that TAK1 is also an important regulator of self-renewal, proliferation, and differentiation of muscle progenitor cells. However, the role and mechanisms of action of TAK1 in RMS remain completely unknown. In this study, we demonstrate that TAK1 expression and activity are markedly elevated in a panel of RMS cell lines and in patient tumor specimens. Reverse phase protein array (RPPA) analyses revealed that TAK1 regulates the expression and activity of many molecules involved in cell cycle control, cell proliferation, and oncogenic signaling. Genetic knockdown or pharmacological inhibition of TAK1 suppresses RMS cell proliferation, migration, and invasiveness, while also promoting terminal myogenic differentiation. TAK1 inhibits differentiation in RMS, at least in part, through up-regulating YAP1 signaling. Our results also demonstrate that inducible knockdown of TAK1 in human RMS xenografts retards tumor growth and enhances myogenic differentiation in vivo. Collectively, these findings uncover a previously unrecognized role for TAK1 in RMS growth and differentiation, and suggest that TAK1 can be a potential therapeutic target for the treatment of RMS.

cancer biology↗

bioRxiv2025

Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

Cerebellar granule cells (GCs) are critical for motor and cognitive functions. Lineage tracing studies have identified a hierarchical developmental progression of GC neurogenesis, transitioning from Sox2+ stem-like cells to Atoh1+ rapidly proliferating granule cell precursors (GCPs), and ultimately to NeuN+ mature GCs. However, the molecular mechanisms governing these transitions remain poorly understood. In this study, we identified a transient, slow-cycling progenitor population defined by co-expression of Sox2 and Atoh1. We show that GC maturation depends critically on the repressive function of the Sin3A/Hdac1 complex, which sequentially silences Sox2 and then Atoh1 to ensure orderly progression through developmental stages. Loss of these repressions prolongs progenitor states, compromises survival, and markedly reduces GC output. We also identify NeuroD1 as a co-repressor that collaborates with Sin3A/Hdac1 to inhibit Atoh1 transcription. Our findings highlight the central role of the Sin3A complex in orchestrating distinct stages of cerebellar GC lineage development and may provide insights into Sin3A-related cerebellar disorders and medulloblastoma in human. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/683101v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f7ebdaorg.highwire.dtl.DTLVardef@19d4cc9org.highwire.dtl.DTLVardef@1c595eorg.highwire.dtl.DTLVardef@12f4b34_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISin3A is sequentially required for GC lineage progression C_LIO_LISin3A promotes the transition of slow-cycling progenitors to GCPs by repressing Sox2 expression C_LIO_LISin3A facilitates GCP differentiation by repressing Atoh1 expression C_LIO_LINeuroD1 recruits the Sin3A/Hdac1 complex to suppress Atoh1 expression C_LI

neuroscience↗

bioRxiv2025

Lake floodplains as sinks for stable soil organic carbon: is the carbon plant- or microbe-derived, and why does permanent land use matter?

Floodplain ecosystems play a key role in soil organic carbon (SOC) storage, as they integrate inputs from both vegetation and sediments. Promoting land uses with low anthropogenic disturbances helps maintain the function of these ecosystems as soil carbon (C) sinks. However, the tipping points along a disturbance gradient where land use transitions generate the largest SOC losses or gains remain unclear, though they are key for effective land use management and climate change mitigation. Because flood events can negatively impact labile C, determining the main origin of stable SOC, whether from plants or soil microorganisms, is also important to identify the optimal combination of land use, vegetation, and soil type for SOC stabilization in floodplains. We examined how SOC storage and stabilization vary in the floodplain of Lake Saint-Pierre, Quebec, Canada along an anthropogenic disturbance gradient of six land uses: conventional and improved croplands, temporary and permanent meadows, marshes, and forested swamps. In all 6 land uses, we quantified SOC stocks, mineral-associated organic matter - C (MAOM-C), particulate organic matter - C (POM-C), soil {delta}13C and sugars. Land use effects on SOC storage and stabilization were most pronounced in the topsoil (0-10 cm), with forested swamps showing the highest vegetation biomass, SOC, and MAOM-C. Microbe-derived inputs were more abundant in MAOM, whereas plant-derived C dominated POM. SOC gains increased with decreasing disturbance, with a tipping point occurring in the transition from temporary to permanent meadows. Our results highlight the importance of conserving permanent and low-disturbance land uses to promote SOC persistence in floodplain ecosystems and emphasize the central role of microbial metabolism in stabilization processes.

ecology↗

bioRxiv2025

Age differences in electrocortical dynamics during uneven terrain walking

Walking on uneven terrain becomes more difficult as we age, and gait becomes less automatic. Using mobile brain imaging via high-density electroencephalography (EEG) can provide insight into the neural mechanisms contributing to reduced mobility capability with aging. The objective of this study was to quantify age differences in electrocortical dynamics during uneven terrain walking, both averaged across many strides and variations within a stride. We included 31 young adults and 71 older adults for analysis. All participants walked on an uneven terrain treadmill with four levels of terrain difficulty at their self-selected speed. Compared to younger adults, older adults exhibited a greater increase in step duration variability and mediolateral sacral excursion variability as the terrain became more uneven. We identified multiple brain regions involved during walking on uneven terrain. Regardless of age group, walking on uneven terrain compared to flat terrain led to a widespread change of electrocortical dynamics in the brain, especially in the alpha (8-13Hz) and beta (13-30Hz) band power. In the parieto-occipital region, younger adults experienced a greater reduction in alpha and beta power with increasing terrain unevenness compared to older adults. We also assessed how intra-stride power fluctuations changed with terrain unevenness and age group. Greater intra-stride power spectral fluctuations in the occipital area were associated with greater terrain unevenness for younger adults, but not for older adults. In summary, older adults showed a greater increase in gait variability than younger adults as the terrain became more uneven, but exhibited a lack of modulation of parieto-occipital activity in response to terrain unevenness. The lack of task-related power modulation may suggest reduced cortical network flexibility in older adults. The absence of increased parieto-occipital activity when walking on uneven versus flat surfaces in older adults may also indicate that, unlike younger adults, older adults already heavily rely on visual processes during flat surface walking and may therefore have reduced occipital modulation range remaining to cope with the visuomotor processing demands of walking on uneven surfaces.

neuroscience↗

bioRxiv2025

Mechanical Cues Regulate Estrogen and Progesterone-Induced Nascent ECM Deposition by Human Endometrial Stromal Cells

The endometrium, the mucosal lining of the uterus, is a highly regenerative tissue that undergoes cyclic remodeling guided by tightly regulated levels of estrogen and progesterone. Stromal cells are embedded within the connective tissue of the endometrium and contribute to the rapidly changing extracellular matrix (ECM). With hormone exposure, endometrial stromal cells undergo decidualization, which alters their morphology and protein secretion. While an increase in tissue modulus is associated with gynecological diseases, the relationship between mechanical properties, hormone exposure, and ECM deposition remains poorly understood. Here, we investigated how both stiffness and hormones regulate ECM deposition by human endometrial stromal cells during decidualization. Using metabolic labeling with sugar analogs and click chemistry, we measure newly secreted ECM proteins deposited by endometrial stromal cells during decidualization. Additionally, we study the nascent ECM in response to different mechanical properties using hyaluronic acid hydrogels. To increase throughput and reproducibility, we designed an automated ImageJ-based workflow for unbiased quantification of nascent ECM deposition. Our results demonstrate that hormones induce decidualization, characterized by F-actin stress fiber formation and prolactin secretion. In addition, we show that decidualization on hydrogels is characterized by an increase in nascent ECM deposition which depends on the initial hydrogel modulus. In contrast, endometrial stromal cells on glass show little change in nascent ECM deposition during hormone exposure. Collectively, these findings demonstrate that both mechanical and biochemical cues regulate ECM deposition during endometrial remodeling. These observations may provide new insights towards future studies addressing the mechanisms of ECM remodeling in gynecological diseases.

bioengineering↗

bioRxiv2025

Season and city shape urban bioaerosol composition beyond vegetation and socioeconomic gradients

Urban vegetation varies with socio-economic gradients, as lower-income neighborhoods often host sparser and less diverse green spaces. This disparity may affect respiratory health by influencing exposure to bioaerosols. Understanding the characteristics of this aerobiome could help anticipate risks related to allergies and other respiratory conditions. Here, we hypothesized that urban vegetation cover and socio-economic status shape urban bioaerosols. We sampled bioaerosols at 65 sites across three Canadian cities of varying population size and density using an active air sampler over four months, and characterized their bacterial, fungal, and pollen composition using amplicon sequencing. Seasonal alpha diversity varied significantly for fungi and pollen. Based on beta diversity, sampling period alone explained up to 40% of pollen, 29% of fungal, and 11% of bacterial community composition variation. In contrast, vegetation cover explained only a minor portion of the variance in bioaerosol composition, and median household income, almost none. These findings provide a critical baseline for understanding the urban aerobiome and highlight the need to study how vegetation identity and diversity, rather than cover alone, may shape bioaerosol dynamics in cities. As cities grow and urban greening initiatives expand, demystifying the aerobiome dynamics becomes an urgent public health priority.

microbiology↗

bioRxiv2025

Responses to Agricultural Practices in Apple Orchards: A Field-Based Mycobiome Study of the Phyllosphere

Microbial communities in the phyllosphere are key players in plant health and disease resistance, yet their response to agricultural management strategies remains poorly understood under field conditions. Here, we compare fungal community composition and diversity across conventional and organic apple orchards using ITS amplicon sequencing. Leaf samples were collected from six sites at three distinct time points during the 2023 growing season (in May, July, and August) corresponding approximately to monthly intervals throughout the summer. Flower samples were collected from the same trees in May. Our analyses reveal that agricultural management strategies are significantly associated with fungal community structure, with effects intensifying from May to July. Both types of management strategies showed enrichment for different genera known to include common apple tree pathogens: Alternaria and Podosphaera were associated with conventional sites, while Didymella and Ramularia were associated with organic sites. Although fungal alpha diversity was higher in May at conventional orchards compared to organic orchards, it declined over time at conventional sites while it remained stable at organic sites. Together, these patterns indicate that distinct management interventions impose contrasting selective pressures on the apple tree phyllosphere mycobiome, thus shaping both broad fungal community composition and the dominance dynamics of key fungal taxa. Our findings underscore the ecological relevance and inherent challenges of field-based microbiome research, and provide insights to inform the development of sustainable orchard management strategies grounded in fungal community dynamics.

microbiology↗

bioRxiv2025

BayesCNet: Bayesian inference for cell type-specific regulatory networks leveraging cell type hierarchy in single-cell data

Understanding gene regulatory networks (GRNs) is essential for deciphering biological processes and disease mechanisms. Single-cell multiome technologies now enable joint profiling of chromatin accessibility and gene expression, offering an powerful means to infer cell type-specific GRNs. However, existing methods analyze each cell type independently or aggregate data into pseudo-bulk profiles, limiting their ability to resolve rare populations and capture cellular heterogeneity. We introduce BayesCNet, a Bayesian hierarchical model that jointly infers enhancer-gene linkages across all cell types while leveraging their hierarchical relationships for information sharing. Through extensive simulations, BayesCNet consistently outperforms state-of-the-art methods, with the largest improvements in rare cell types. When applied to real datasets, BayesCNet identifies enhancer-gene linkages with higher accuracy validated by promoter-capture Hi-C data, and reconstructs cell type-specific GRNs that highlight key regulators, demonstrating its power to resolve gene regulatory programs across diverse cell types.

bioinformatics↗

bioRxiv2025

Diverse epigenomic mechanisms underpin transcriptional dysregulation in Polycomb-altered acute myeloid leukemia

BackgroundPolycomb Repressive Complex 2 (PRC2) modulates chromatin accessibility and architecture to direct tissue-specific gene expression. PRC2 function is frequently altered in cancer by loss-of-function mutation or deletion, but the downstream effects on transcriptional regulation are incompletely understood. ResultsTo gain insights into these mechanisms, we performed a holistic analysis of epigenomic and transcriptional changes in an isogenic model of acute myeloid leukemia (AML) with heterozygous EZH2 deletion that mimics reduced PRC2 function in patient leukemias. PRC2-depleted cells had diverse gene expression changes, including a bias towards more immature monocyte-lineage transcriptional signatures. PRC2 depletion also correlated with marked increases in chromatin accessibility genome-wide, with 10-45% increases in ATAC-seq peaks in EZH2+/- clones. These changes were accompanied by decreased H3K27me3 and increased H3K27ac levels in CUT+RUN assays that were incompletely linked to transcriptional activity. Despite these generalised changes, 3D chromatin architecture assessed by Hi-C was largely maintained, with H3K27me3 preferentially lost in regions with low DNA-DNA contact frequency. Surprisingly, some regions gained broad H3K27me3 domains at heavily compacted chromatin. We notably saw compartmentalisation changes upstream of the transcriptionally upregulated fetal hematopoiesis gene LIN28B in EZH2+/- cells, with corresponding activation of a LIN28B-specific transcriptional program, including upregulation of the CDK6 oncogene. These results correlated with EZH2+/- cell phenotype, including decreased cellular proliferation and increased resistance to CDK6 inhibitor palbociclib. ConclusionsOur findings suggest that PRC2 depletion pleiotropically affects AML transcriptional regulation to directly impact cell phenotype and treatment responsiveness, which may partially explain the aggressive biology seen in these cases.

cancer biology↗

bioRxiv2025

Non-invasive brain stimulation protects cognitive impairment in i.c.v.STZ injected rats: role of adult neurogenesis

In Alzheimers like dementia, neurodegeneration and synaptic dysfunction is known to the critical player in explaining the cognitive impairment. Adult neurogenesis, normally a chronic and quite process is explained to have potential in the field of Alzheimers therapy. Previous research on the non-invasive brain stimulation showed that controlling pattern of stimulation externally we can regulate/ entrain neuronal activity, possibly altering the structural changes in the circuit level. However, literature investigating if non-invasive brain stimulation could hold any potential to initiate process of adult neurogenesis are scarce. In the present study, with the use of behavioural, microscopic and biochemical tools we found that extremely low frequency magnetic field at an intensity of 17.96{micro}T with sinusoidal wave of 50Hz for 2hr daily for a period of two week in streptozotocin induced animal model of sporadic Alzheimers disease can cause improvement in spatial and reference memory, influencing their swimming strategy in water maze. Which is caused by stimulation in immature neural pluripotent stem cells, with additional redox balance and mitigation of glial aggravation in brain areas like olfactory bulb, prefrontal cortex and hippocampus. These changes were accompanied by neuroprotection as observed in the granular layer of dentate gyrus. Taken together, present study explains plausible mechanism of action on protection of neurodegeneration in Alzheimers disease through non-invasive brain stimulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/666022v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@bc7830org.highwire.dtl.DTLVardef@176cc01org.highwire.dtl.DTLVardef@4b869dorg.highwire.dtl.DTLVardef@72e703_HPS_FORMAT_FIGEXP M_FIG Graphical illustration representing external magnetic field causing cognitive improvement eliciting adult neurogenesis in animal model of Alzheimers disease C_FIG

neuroscience↗

bioRxiv2025

Ribonucleoprotein (RNP) condensates modulate survival in response to Mars-like stress conditions

Scientific advances have emboldened human efforts toward exploring the potential of extinct, extant, or future life on Mars. An important aspect of this endeavor is understanding how an organism adapts to stress-inducing environmental conditions on Mars, such as radiation, shock waves, extreme temperatures, and chaotropic stress due to higher levels of perchlorates. A conserved approach used by organisms across evolutionary scales to adapt and overcome stress conditions is the assembly of ribonucleoprotein (RNP) condensates. In this study, we employ a multidisciplinary approach to understand yeast survivability and adaptation under Mars-like stress conditions, specifically shock waves and perchlorate, by focusing on RNP condensates. Our study reveals that yeast survives 5.6 M intensity shock waves. Exposure to either shock waves or sodium perchlorate induces the formation of P-bodies, a conserved stress-induced condensate. Yeast mutants defective in P-body assembly show defective growth in response to perchlorate stress. Transcriptome analysis, followed by validation, identified several relevant transcripts whose levels are perturbed in response to Mars-like conditions. Finally, identification of several transcripts whose abundance is altered in the P-body assembly mutant upon stress highlights a new connection between response to Martian stress conditions and RNP condensates. This study, a first of its kind, highlights the importance of RNP condensates in understanding the impact of Martian conditions on life in general. This study paves the way for using RNP condensates as a biomarker for assessing the health of life forms during space explorations.

cell biology↗

bioRxiv2025

Molecular insights into inhibitor action on a key bacterial metabolic enzyme Cystathionine β-Synthase

Tuberculosis (TB) remains a major global health threat, with Mycobacterium tuberculosis (Mtb) infecting nearly a quarter of the global population. Drug-resistant TB and HIV-TB co- infections emphasize the need for novel therapeutic approaches targeting essential metabolic pathways. Here, we investigated Mtb cystathionine {beta}-synthase (MtbCBS), a PLP-dependent enzyme critical for sulfur metabolism and redox regulation, owing its potential as a therapeutic target. We present the first high-resolution cryo-EM structure of MtbCBS bound to aminooxyacetic acid (AOAA), and employed molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, and comparative inhibition studies to reveal the molecular basis and determinants governing irreversible PLP-enzyme inhibition. Our Cryo-EM structural analysis revealed two highly conserved active-site residues, T75 and Q147, critically stabilizing the inhibitor complex. Through molecular mimic studies, we demonstrated the precise structural factors and electronic features critical for inhibition efficiency. These findings provide the first mechanistic rationale for PLP enzyme inhibition and offer a generalizable framework for designing covalent inhibitors targeting PLP-dependent enzymes implicated in infectious diseases, cancer, and neurological disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/662948v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@195c7c7org.highwire.dtl.DTLVardef@6ee0c8org.highwire.dtl.DTLVardef@1679dfborg.highwire.dtl.DTLVardef@1b4e000_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Proposed mechanism of MtbCBS inhibition by AOAA. Proposed mechanism of action of AOAA illustrated through a graphical representation. The cartoon shows the domains of MtbCBS. Tetrameric structure of MtbCBS with active site highlighted (in red dashes). Enlarged view indicating the PLP-AOAA blocked intermediate interacting with residue Q147. Reaction scheme depicting the previous missing understanding of active site residue role in -elimination. Schematic representation of AOAA inhibition mechanism in the catalytic cycle of MtbCBS. Reactions were drawn using ChemDraw. The proposed scheme highlights that AOAA (highlighted Red) binding is stabilized by nearby residues Q147 and T75, leading to the formation of PLP-AOAA covalent adduct, and substrates (highlighted green) were incapable of reverting the suicidal inhibition. The directionality and reversibility were mentioned for each step. C_FIG

biophysics↗

bioRxiv2025

DTH: A nonparametric test for homogeneity of multivariate dispersions

Testing homogeneity across groups in multivariate data is an important scientific question in its own right, as well as well as an auxiliary step in verifying the assumptions of ANOVA. Existing methods either construct test statistics based on the distance of each observation from the group center, or as the mean of pairwise dissimilarities among observations in a group. Both approaches can fail when mean within-group distance is similar across groups but the distribution of the within-group distances are different. This is a pertinent question in high dimensional microbiome data, where outliers and overdispersion can distort the performance of a mean-dissimilarity-based test. We introduce the non-parametric Distance based Test for Homogeneity (DTH) which measures dissimilarity between groups by comparing the empirical distribution of within-group dissimilarities using a combination of the Kolmogorov-Smirnov and Wasserstein distances. For more than two groups, pairwise group tests are combined using a permutation-based p-value. Through simulations we show that our method has higher power than existing tests for homogeneity in certain situations and comparable power in other situations. We also provide a simple framework for extending the test to a continuous covariate.

genomics↗

bioRxiv2025

Hydrogel viscoelasticity modulates cell nascent extracellular matrix deposition

Polymeric hydrogels are valuable platforms for determining how specific mechanical properties of native tissue extracellular matrix (ECM) regulate cell function. Recent research has focused on incorporating viscous and elastic properties into hydrogels to investigate cellular responses to time-dependent mechanical properties of the ECM. However, a critical aspect often overlooked is that cells continuously remodel their microenvironment in hydrogels, such as by the deposition of newly secreted (nascent) ECM. While this nascent ECM has been demonstrated to play a vital role in transmitting mechanical signals across various biological contexts, the mechanisms by which it regulates cellular function in response to time-dependent mechanical properties remain poorly understood. In this study, we developed an interpenetrating polymer network that enables independent control of viscous and elastic hydrogel properties. We show that cells cultured on high-viscosity hydrogels deposit increased nascent ECM which also correlates with enhanced hydrogel remodeling. Interestingly, higher nascent ECM deposition on high-viscosity hydrogels was decoupled from intracellular contractility. These results establish a relationship between hydrogel viscosity and nascent ECM deposition that may extend to diverse cell types and offer new insights into cell-hydrogel interactions.

bioengineering↗