Biology subjects

Datta, S.

Publications and source records attributed to Datta, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Effect of Varying Cigarette Smoke Concentrations and Durations on the Retinal Pigment Epithelium

Dry age-related macular degeneration (AMD) is a leading cause of blindness in the elderly, and treatment options are limited. Cigarette smoke (CS), the primary modifiable environmental risk factor for AMD, damages the retinal pigment epithelium (RPE) by inducing oxidative stress, which in turn activates multiple stress response pathways. Low levels of stress induce autophagy, a cell-recycling mechanism; moderate stress induces epithelial-mesenchymal transition (EMT); and high, sustained stress, once other cytoprotective pathways are exhausted, leads to cell death. All three pathways are implicated in AMD pathogenesis. Although CS is known to induce oxidative stress, its impact on these distinct stress response pathways remains understudied, and few studies have directly compared mouse and human RPE responses to the same stressor. Here, we examined the effect of varying concentrations and durations of cigarette smoke condensate (CSC) exposure on oxidative stress, autophagy, EMT, and cell death in ARPE-19 (human) and mouse RPE (MRPE) cells. Understanding which stress response pathways predominate at which CSC exposure levels may help guide the development of AMD treatments stratified by smoking history.

cell biology↗

bioRxiv2026

Cryo-EM structures of the YsaN ATPase reveal dodecameric and tetradecameric assemblies in the Yersinia enterocolitica T3SS

Bacterial pathogens deploy sophisticated nanomachines such as the Type III Secretion System (T3SS) to inject effector proteins directly into host cells, thereby subverting cellular defenses and promoting infection. Central to this machinery is a conserved homo-oligomeric ATPase that fuels substrate unfolding and translocation. Despite extensive structural and biochemical studies, the precise mechanism of T3SS ATPase oligomer assembly and regulation remains incompletely understood, events crucial for the overall functionality of the T3SS. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the Yersinia enterocolitica T3SS ATPase YsaN, captured in two previously uncharacterized oligomeric states, a dodecamer (12-mer) at 3.51 [A] and a tetradecamer (14-mer) at 3.80 [A] resolution. Both assemblies exhibit helical-like symmetry and conformational heterogeneity. A conserved longitudinal fissure interrupts the helical lattice and appears to mediate interconversion between the two states, while an N-terminal interface stabilized by inter-tier disulfide bonds and an extensive non-covalent network locks the assembly together. Molecular dynamics simulations reveal that ATP hydrolysis drives a global, correlated compaction upon transition to the ADP-bound state. Together, these structural, biochemical, and MD simulation data establish T3SS ATPases as dynamic, oligomerically plastic molecular motors and provide a mechanistic framework for chaperone-effector dissociation and substrate translocation.

biophysics↗

bioRxiv2026

Metabolic disease-relevant stimuli unmask context-dependent genetic regulation of cardiometabolic loci in human adipocytes

Genome-wide association studies (GWAS) have identified thousands of loci associated with cardiometabolic disease, yet translating these associations into regulatory mechanisms, effector genes, and cellular programs remains a major challenge. A key limitation is that genetic effects are often highly context dependent, varying across cell states and environmental conditions that are difficult to model at scale. Here, we leverage CellGenBank, a population-scale biobank of primary human adipose-derived mesenchymal stem cells (AMSCs), to implement a multi-donor cell village in vitro system to map cardiometabolic disease genetic variation across adipocyte differentiation and metabolic stress conditions. We pooled AMSCs from 118 donors into multiplexed villages, differentiated them toward adipocytes, and profiled chromatin accessibility and gene expression using single-nucleus multiome sequencing under four disease-relevant conditions: basal, elevated free fatty acids, low glucose, and hypoxia. By combining with genetic demultiplexing, we quantify how regulatory element activity, gene expression, and higher-order cellular programs are modulated by both genotype and environmental context. Across conditions, we identify widespread context-specific cis-regulatory effects, including expression and chromatin accessibility quantitative trait loci that are masked in baseline states. Genetic effects frequently converge on coordinated transcriptional programs linked to lipid metabolism, insulin responsiveness, and stress adaptation, enabling the identification of cellular program QTLs that bridge variants, genes, and disease-relevant phenotypes. Integration with cardiometabolic GWAS reveals enhanced colocalization in condition- and state-resolved analyses, highlighting the importance of modeling environmental context to resolve disease mechanisms. Together, our study establishes large-scale adipocyte cell villages as a powerful and generalizable framework to map the context-dependent regulatory architecture of cardiometabolic disease and provides a resource linking human genetic variation to adipocyte cellular programs.

genomics↗

bioRxiv2026

Surface Binding Sites Orchestrate Long-Range Control of Active-Site Dynamics and Glucose Tolerance in β-glucosidase, BglB

{beta}-Glucosidases are essential for lignocellulosic biomass conversion, yet their industrial utility is limited by inhibition from glucose, the reaction product. Although glucose inhibition has been extensively documented, its molecular basis remains poorly understood. Here, we demonstrate that glucose inhibition in the GH1 {beta}-glucosidase BglB from Paenibacillus polymyxa is mediated by multiple surface-exposed secondary binding sites (SBSs) that function as regulatory elements rather than passive glucose-binding patches. Kinetic analyses revealed a mixed mode of inhibition, with modest activation at low glucose concentrations followed by progressive inhibition at higher concentrations. Glucose association occurred through multiple SBSs in a concentration-dependent manner and induced localized rigidification, particularly at gatekeeper regions surrounding the active-site entrance. These surface interactions propagated through long-range coupling pathways, resulting in structural, energetic, and residue interaction network reorganization that reshaped the catalytic pocket. Functional interrogation of representative SBSs revealed distinct roles for individual sites. Mutation of a gatekeeper-associated SBS reduced local glucose association, increased glucose tolerance by more than 30%, and improved substrate affinity by approximately 38%, whereas disruption of a distal SBS compromised structural integrity and soluble protein production. Together, these findings establish a direct link between surface glucose recognition and active-site regulation, providing, to our knowledge, the first integrated evidence for the existence and functional significance of secondary glucose-binding sites in {beta}-glucosidases. More broadly, this work identifies surface SBSs as promising targets for engineering glucose-tolerant and catalytically robust enzymes for biomass conversion and related biotechnological applications.

biophysics↗

bioRxiv2026

Cell Mechanics Regulate Membrane Tubulation-Driven Trogocytosis in Adherent Cells

Trogocytosis is a widespread and physiologically important process in which one cell ingests fragments of another. It has been observed in diverse biological contexts, such as in Entamoeba histolytica, a human enteric parasite that nibbles host intestinal cells via trogocytosis, thereby invading the tissue and leading to lethal extra-intestinal disease, and in macrophages on interaction with adherent tumor monolayers. Despite its physiological relevance, knowledge of trogocytosis with adherent cells remains elusive. Here, we established a monolayer-based trogocytosis assay and uncovered a distinct membrane-tube-mediated trogocytic mechanism operative in adherent cells. We characterized it into four sequential events: contact, membrane tubulation, stretching, and scission. By targeting the proteic constituents of target cell stiffness, we show that trogocytic output exhibits a non-monotonic dependence on the viscoelastic properties of the target cell, with maximal uptake occurring in an intermediate mechanical regime. Further, we integrated live-cell trogocytosis observations with a viscoelastic model-based theoretical framework to propose a tube-breaking mechanism across distinct mechanical subtypes. Finally, using micropatterning, we demonstrated that target cell shape influences trogocytosis kinetics. This work suggests that trogocytosis is sensitive to the mechanical state of the target cell.

cell biology↗

bioRxiv2026

Osteocyte Perilacunar/canalicular Remodeling (PLR) Drives Spatially Heterogeneous Lacunar Remodeling During and After Lactation

BackgroundPregnancy and lactation impose substantial demands on maternal calcium homeostasis, leading to pronounced skeletal remodeling during lactation followed by recovery after weaning. Although bone mass is largely returned at the tissue level after weaning, it remains unclear whether osteocyte-level remodeling exhibits a similarly reversible pattern. Osteocytes regulate mineral mobilization through perilacunar/canalicular remodeling (PLR), which is elevated during lactation. However, its spatial and temporal regulation in response to reproduction remains poorly defined. Objective/HypothesisThis study aims to determine whether PLR-regulated lacunar remodeling during reproduction varies with osteocyte location and relative age. We hypothesized that osteocyte PLR-mediated lacunar change is spatially heterogeneous during lactation, varies with osteocyte location and relative age, and may persist after weaning. MethodsFemale wild-type (WT) and osteocyte-specific PTH/PTHrP receptor knockout (cKO) mice were studied across reproductive stages. Longitudinal in vivo DXA and CT were used to assess skeletal changes. Sequential fluorochrome labeling tracked mineral dynamics and defined osteocyte relative age. Osteocyte PLR activity was evaluated by MMP13 immunohistochemistry. Lacunar-canalicular structure (LCS) was assessed using Ploton silver nitrate staining, and spatially resolved lacunar morphology was quantified using high-resolution backscattered scanning electron microscopy (bSEM). ResultsAt the tissue level, reproduction induced distinct skeletal responses, characterized by reversible cortical bone loss and persistent trabecular deterioration. Cortical bone loss during lactation was spatially asymmetric and confined to the posterior cortex. Fluorochrome labeling further resolved surface-specific remodeling patterns during reproduction, including endocortical resorption at the posterior cortex and sustained deposition at anterior endocortical and posterior periosteal surfaces. At the cellular level, osteocyte PLR activity increased during lactation in WT mice and returned after weaning, whereas no changes were observed in cKO mice. Consistently, lacunar size increased during lactation and returned toward baseline after weaning in WT mice but remained unchanged in cKO mice. Spatially resolved analysis demonstrated that lacunar remodeling was heterogeneous across cortical thickness. At the anterior cortex, lacunar enlargement occurred near the endocortical surface during lactation and was reversible after weaning. In contrast, at the posterior cortex, lacunar enlargement occurred near the periosteal surface and persisted after weaning. These spatial patterns corresponded to cortical regions enriched with newly formed osteocytes, whereas pre-existing osteocytes exhibited minimal changes. This spatial heterogeneity was absent in cKO mice. ConclusionOsteocyte PLR-mediated lacunar remodeling during reproduction is spatially heterogeneous and varies with osteocyte location and relative age. These findings demonstrate that recovery at the tissue level does not necessarily extend to the osteocyte microenvironment and identify osteocyte PLR-mediated lacunar remodeling as a spatially structured and potentially persistent component of reproductive skeletal adaptation. Together, these results highlight a previously unrecognized layer of maternal skeletal health.

bioengineering↗

bioRxiv2026

Dietary serine protects the retinal pigmented epithelium by blunting reactive oxygen species in dry age-related macular degeneration

Age-related macular degeneration (AMD) is a progressive complex eye disease and one of the leading causes of blindness. AMD progression is marked by molecular changes in the retinal pigmented epithelium (RPE) which include increased reactive oxygen species (ROS) accumulation, mitochondrial dysfunction - eventually leading to dysfunctional RPE. Mitophagy regulator, Pink1, is reduced in the RPE of AMD patients and Pink1 loss leads to a shift from mitochondrial respiration to glycolysis. Serine is a non-essential amino acid which is de novo synthesized from glycolytic intermediate 3-PG via the rate limiting enzyme PHGDH. Serine is tightly integrated into anabolic processes like glutathione (GSH) cycling, maintaining NADH/NADPH pools leading to changes in AMPK signaling. Here, we show that Pink1 loss leads to a reduction in PHGDH and serine levels in the RPE leading to impaired mitochondrial structure and function, increased ROS mediated damage, increased inflammation, and hampered retinal function. Serine supplementation rescued ROS accumulation, balanced GSH abundance, and increased retinal function. Overall, our study highlights the potential of dietary serine in ROS management in AMD.

molecular biology↗

bioRxiv2026

Engineering a Glucose-Inducible Whole-Cell Biosensor via CRISPRi-Based Promoter Reprogramming

Precise monitoring of intracellular glucose dynamics is essential for understanding carbon flux, optimizing microbial bioprocesses, and enabling responsive control of engineered metabolic pathways. Here, we develop a modular whole-cell biosensor in Escherichia coli that converts the native glucose repression phenotype of a CAP-sensitive promoter into a tunable, glucose-inducible output using CRISPR interference (CRISPRi). By placing a guide RNA (gRNA) under the control of the CAP promoter and positioning dCas9 to target the -10 region of a constitutive promoter driving sfGFP, we created an inversion circuit in which glucose suppresses gRNA expression, thereby relieving dCas9-mediated repression and activating fluorescence. Systematic evaluation of gRNA strand orientation and target site selection revealed that template-strand targeting yielded strong repression ([~]90 %) but reduced sensing range, whereas moderately repressive non-template gRNAs ([~]27-35 % repression) enabled optimal signal inversion. The resulting biosensor demonstrated a robust, linear fluorescence response across 200 M -50 mM glucose (R{superscript 2} > 0.97), with high specificity against other sugars and a strong correlation between glucose consumption and fluorescence accumulation (R{superscript 2} {approx} 0.996). To extend the functionality of the platform, we integrated the sensor with a secreted {beta}-glucosidase module that hydrolyzes cellobiose to glucose. The biosensor accurately quantified glucose released during cellobiose degradation, with engineered strains producing up to [~]33 mM glucose from 50 mM cellobiose in a two-plasmid system. This coupling of enzymatic conversion with intracellular sensing enabled real-time, non-destructive monitoring of metabolic transitions. Together, this work establishes a programmable CRISPRi-based strategy for inverting native promoter logic and provides a sensitive, specific, and modular platform for metabolite sensing in bacteria. The approach is broadly applicable for dynamic pathway regulation, monitoring carbon fluxes, and building responsive genetic circuits in metabolic engineering and synthetic microbial ecosystems. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/717388v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@158eb53org.highwire.dtl.DTLVardef@c1a21eorg.highwire.dtl.DTLVardef@7a87f7org.highwire.dtl.DTLVardef@746e80_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

Retinal Pigment Epithelium Injury in Pentosan Polysulfate Exposure: Morphologic Changes, Phagocytic Deficits, and Mitochondrial Dysfunction

Pentosan polysulfate (PPS) is a semisynthetic sulfated polysaccharide that was approved by the United States Food and Drug Administration (FDA) for treatment of interstitial cystitis (IC). A 2018 study by our group described a vision-threatening macular toxicity associated with long-term use of PPS. However, given the relatively recent characterization of PPS maculopathy, we have limited knowledge of its pathophysiology. The present study therefore investigated the pathophysiology of PPS maculopathy in a cell culture model, assessing impacts of PPS exposure on morphology and mitochondrial function. We treated ARPE-19 cells with increasing doses of PPS and investigated both mitoprotective and cytoprotective mechanisms, mitochondrial reactive oxygen species production (ROS) and respiration, cellular structure, and retinal pigment epithelium (RPE) dysfunction through phagocytosis assays. We found that PPS increased mitochondrial superoxide accumulation and that increased doses of PPS impaired basal and maximal respiration in a Seahorse assay without the expected response of increases in the cellular energy sensor pAMPK. PPS exposure disrupted mitochondrial and cell protective mechanisms against ROS accumulation as assessed through examination of mitochondrial biogenesis markers PGC-1 and SIRT1 and autophagy markers LC3 and p62. PINK1 expression increased with increasing duration of exposure to PPS. Further, we found that PPS led to functional and structural changes to RPE cells, which exhibited an increase in cell aspect ratio and impaired phagocytosis with higher doses of PPS. Lastly, we found an increase in cell death in response to higher doses of PPS, evident through ethidium homodimer cell viability assays. Taken together, our study shows PPS exposure has profound effects on RPE viability and function through impairment of mitochondrial respiration and mito- and cyto-protective mechanisms and highlights mitochondrial insult as a potential focus of future PPS research.

molecular biology↗

bioRxiv2026

Deciphering sepsis molecular subtypes using large-scale data to identify subtype-specific drug repurposing

Sepsis is a life-threatening dysregulated response to infection, the heterogeneity of which precludes effective targeted therapies. To address this, we created a transcriptomic atlas of publicly available adult sepsis data, on which we performed molecular subtyping and identified potential subtype-specific drug repurposing opportunities. In total, we harmonized data from 3,713 samples across 28 datasets, of which 2,251 were from sepsis patients. Using this data, we identified four molecular subtypes of sepsis (C1 - C4) by clustering the sepsis samples based on expression differences in immune-and lipid-related genes. We next identified gene signatures unique to each molecular subtype. Pathway analysis of these signatures revealed patterns of immune exhaustion and metabolic dysregulation in C1, suggesting potential benefit from corticosteroid treatment. C2 had the youngest patient population and the lowest mortality, and C2 expression patterns were often anti-correlated with those of C1. C3 was enriched for inflammatory and cellular stress pathways, while the highest mortality subtype, C4, showed evidence of immunosuppression and metabolic reprogramming. Gene and pathway-level analysis of our molecular subtypes statistically correlated with results from analysis of 28-day mortality, with the best (C2) and worst subtypes (C4) exhibiting similar molecular dysregulation as survivors and non-survivors, respectively. For each subtype, we then evaluated potential targeted therapies. Using a large-scale pharmacogenomics database, we identified drugs targeting the subtype gene signatures and assessed the potential clinical impacts of these drugs. We identified several potential candidate therapies for each molecular subtype, including possible responsiveness to Methylene Blue therapy for patients from our highest mortality subtype, C4. Notably, our drug repurposing analysis revealed a significant representation of anti-inflammatory monoclonal antibody therapies across molecular subtypes. The anti-correlated signatures in C1 and C2 suggest that monoclonal antibody therapies may not be effective for patients in both subtypes, which may explain why prior clinical trials have been unsuccessful. Altogether, our detailed molecular subtyping and analysis identify potential drug targets within each molecular subtype, with implications for future precision medicine for sepsis.

bioinformatics↗

bioRxiv2026

From genes to collective modes: biological constraints shape metabolic evolution

Population genetics has been successful in explaining how drift, selection, and mutation shape allele frequencies. However, we still lack an understanding of how biological and evolutionary constraints arising from genotype-phenotype maps shape evolutionary dynamics. To explore this question, we developed a new framework for simulating the evolution of an inherently polygenic and epistatic trait - metabolism - combining population genetics with flux balance analysis. We found that the evolution of metabolic networks exhibits surprisingly simple, reproducible dynamics. We identify evolutionary collective modes (EvCMs) - linear combinations of genes with high, constant selective pressure - as organizers of the long-term dynamics and the origin of this simplicity. EvCMs arise through the interaction of physical constraints, evolvability, and the requirements for growth. We developed a theoretical framework to predict EvCMs from large-scale metabolic models and found quantitative agreement between theory and simulation of E. coli central metabolism. Inspired by these results, we re-analyzed mutational data from Lenskis long-term evolution experiment and found compelling evidence for the existence of EvCMs. This work suggests that biological constraints encoded in genotype-phenotype maps play an important role in shaping evolutionary dynamics, offering a new perspective on complex trait evolution -- one that shifts focus from individual genes to collective modes.

evolutionary biology↗

bioRxiv2026

Classical enhancers couple cis-regulatory logic with transcriptional condensates and 3D genome architecture

Deciphering the regulatory logic of enhancers remains a central question in understanding cell- and tissue-specific gene expression in multicellular organisms. This is particularly pertinent at multipartite enhancer clusters such as super-enhancers, where multiple enhancers contribute to the expression of a single gene. Gene expression has been studied largely through sequence-dependent recruitment of transcription factors (TF) and co-activators, whereas 3D chromatin structure has been attributed to architectural proteins such as cohesion and CTCF1-3. However, the contribution of DNA sequence encoded in enhancers to shaping higher-order genome organization remains poorly understood. Here we show that classical enhancers, embedded within multipartite super-enhancer structures, act as determinant regulatory elements that initiate the gene regulatory cascade by linking DNA sequence recognition to 3D chromatin architecture. Classical enhancers are more evolutionarily conserved and display stronger regulatory activity than facilitator elements, which lack intrinsic enhancer activity but potentiate classical enhancer function. We show that classical enhancers are selectively bound by specific TFs with strong intrinsically disordered regions, such as NFE2L2 in liver cancer cells, capable of driving transcriptional condensate formation through phase separation. NFE2L2 depletion reduced enhancer activity and induced widespread chromatin reorganization, characterized by increased cohesin and CTCF occupancy at super-enhancer boundaries and beyond. This "cohesin clogging" impaired DNA loop extrusion, led to formation of smaller topologically associated domains, and weakened enhancer-promoter contacts. These findings highlight that sequence-specific TFs have multifaceted roles beyond transcriptional control, establishing a direct mechanistic link between enhancer sequence, TF binding, condensate formation, and 3D genome organization, with the regulatory logic being encoded in the DNA sequence itself.

genomics↗

bioRxiv2026

Prime editing of an evolutionarily informed PEPC residue enhances photosynthesis and grain nutrition in C₃ rice

C4 photosynthesis evolved through adaptive changes in key metabolic enzymes that enhance carbon fixation efficiency, yet transferring such traits into C3 crops has met limited success. Using precision prime editing, we introduced an evolutionarily conserved single amino acid substitution (Arg[->]Gly) into phosphoenolpyruvate carboxylase (PEPC) in rice. Edited plants exhibited enhanced PEPC activity in the presence of the feedback inhibitor malate, increased chlorophyll content, elevated photosynthetic rates under ambient CO2, and increased seed size and weight. Remarkably, seeds from the edited lines showed simultaneous enrichment of zinc (Zn), iron (Fe), and protein, hereafter referred to as ZiP rice. This work demonstrates that a single, evolutionarily guided residue change can reprogram core carbon metabolism in a C3 crop, enabling concurrent improvements in photosynthesis and grain nutritional quality.

plant biology↗

bioRxiv2025

A Highly Thermostable and Novel GH5 Endoglucanase from Bacillus sp. Strain BS with Enhanced Biomass Saccharification Potential in Seawater

The efficient enzymatic conversion of lignocellulosic biomass to glucose is crucial for biofuel production, with enzyme costs being a significant barrier to commercialization. Multifunctional enzymes that reduce enzyme components can improve efficiency. In this study, we amplified and sequenced a gene from Bacillus sp. strain BS, which showed 89.8% identity with bacterial endoglucanase Q6YK34. This putative novel endoglucanase, BsEG2, is a GH5-like endoglucanase, with optimal activity at pH 6.0 and 55 {square}, and efficiently hydrolyzes amorphous and crystalline cellulose, including pre-treated and untreated sugarcane bagasse. Kinetics on carboxymethyl cellulose (CMC) indicate high substrate binding efficiency (apparent Vmax of 148.9 {micro}M min-1, a Km of 24.9 mg mL-1, and a kcat of 517 s-1). BsEG2 hydrolyzes long cellulosic chains, oligosaccharides, and cellobiose to produce glucose, demonstrating multifunctionality. It exhibits remarkable thermal stability, retaining over 90% activity after 15 days at 55 {degrees}C. BsEG2 also retains activity in high salts and ionic liquids, and is not inhibited by cellobiose, even at 200 mM, unlike other endoglucanases and cellobiohydrolases. Furthermore, BsEG2 functions in seawater, reducing freshwater footprint in biofuel production. BsEG2 is a promising candidate for a cost-effective enzyme formulation for glucose production from lignocellulosic biomass, supporting downstream glucose-fed metabolic pathways.

biochemistry↗

bioRxiv2025

Antagonism between blue and red light-signalling controls thallus flatness in Marchantia polymorpha

The growth orientation of the Marchantia polymorpha thallus - a system of dorsiventralized, indeterminate axes - is modulated by light. We show that red and blue light act antagonistically to control thallus growth tropisms, with red light signalling promoting epinasty and blue light signalling promoting hyponasty. We found that loss-of-function mutations in the blue light receptor MpPHOT led to epinasty, while loss-of-function mutations in the red light receptor MpPHY resulted in hyponasty. We hypothesize that these antagonistic activities of blue and red light signalling are balanced in white light, resulting in the development of flat thalli. Using time-resolved transcriptomics, we identified genes that were rapidly induced upon light exposure. Among these genes were all six members of the M. polymorpha BBX gene family. Mutants harbouring loss-of-function mutations in two of the six MpBBX transcription factors developed defective thallus tropisms. Mpbbx1 loss-of-function mutants formed hyponastic thalli, while Mpbbx5 loss-of-function mutants developed epinastic thalli. Double mutants Mpbbx1 Mpbbx5 grew flat, supporting the hypothesis that they function antagonistically. Together, these data indicate that phototropin-mediated blue light and phytochrome-mediated red light signalling antagonistically modulate thallus tropism, and that BBX transcription factors also act antagonistically to regulate thallus flatness.

plant biology↗

bioRxiv2025

RAB14-dependent tubulovesicular recycling directs MET to invadopodia promoting TNBC cell invasion

Metastasis is one of the primary causes of cancer-related death in Triple-negative breast cancer patients. During metastatic dissemination, the cancer cells infiltrate in to surrounding tissue employing specialized membrane-protrusion with proteolytic activity called invadopodia. Cues from growth factors via the cognate receptor tyrosine kinases promote invadopodia formation and cancer cell invasion. We report the role of HGF and its cognate receptor MET in TNBC invasion. The results from our study demonstrate that the TNBC cells when stimulated with HGF promotes invadopodia formation and facilitates delivery of MT1-MMP to the invadopodia in a MET dependent manner. We also showed that MET resides at invadopodia and its localization at invadopodia increases upon HGF treatment due to enhanced surface delivery of the RTK. By employing a degradation defective mutant, we demonstrated the critical role of MET recycling in driving breast cancer invasion. Moreover, we observed that the RCP-RAB14 axis mediates the surface delivery of MET through tubulovesicular carriers, with KIF16B promoting the formation of vesicular tubules on RAB14-positive endosomes. Collectively, the study provides mechanistic insights on the growth factor-mediated recycling of RTK and the associated metalloprotease in invadopodia-dependent matrix degradation and cancer cell invasion.

cancer biology↗

bioRxiv2025

Domain Compatibility and Linker Design Dictate the Success of Chimeric Cellulase Engineering

Efficient conversion of lignocellulosic biomass into fermentable sugars remains a major challenge due to individual cellulases limited synergy and catalytic efficiency. Engineering chimeric enzymes provides a promising strategy to streamline biomass hydrolysis by combining complementary catalytic activities in a single protein, thereby enhancing efficiency and lowering process costs. In this study, we constructed chimeric cellulases by fusing a thermophilic GH1 {beta}-glucosidase (TsBG) with endoglucanases from the GH5 (BsEG2) or GH9 (BlEG) families through flexible peptide linkers. Constructs containing BsEG2 exhibited a pronounced loss of {beta}-glucosidase activity and reduced endoglucanase activity, whereas substitution with the full-length BlEG restored dual functionality under identical design conditions. The optimized chimera (BlEG+(G4S)2+TsBG) demonstrated enhanced catalytic performance, with a 4.8-fold lower Km, a 1.7-fold higher Vmax, and an increased kcat (from 1088 to 1454 s-1). The chimera also exhibited enhanced stability, retaining [~]10 % higher activity under elevated cellobiose (up to 300 mM) and >90 % specific activity in 2.5 M NaCl. Molecular dynamics simulations further revealed that activity loss in non-optimized constructs arose from C-terminal structural instability and steric clashes, underscoring the critical role of domain orientation and linker flexibility in chimera design. These findings establish a chimeric cellulase that integrates endoglucanase and {beta}-glucosidase activities in a single polypeptide, offering a robust and cost-effective biocatalyst for lignocellulosic biomass conversion.

biochemistry↗

bioRxiv2025

Real-time, High-throughput Super-resolution Microscopy via Panoramic Integration

We introduce super-resolution panoramic integration (SPI), an on-the-fly microscopy technique enabling instantaneous generation of subdiffractional images concurrently with scalable, high-throughput screening. SPI leverages multifocal optical rescaling, high-content sweeping, and synchronized line-scan readout while preserving minimal post-processing and compatibility with epi-fluorescence settings. We demonstrate SPI for various subcellular and populational morphology, function, and heterogeneity. This versatile platform offers a practical pathway toward biological insights beyond traditional optical and computational constraints.

bioengineering↗