Biology subjects

Paul, D.

Publications and source records attributed to Paul, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Mechanistic insights into proteolytic activation ofmembrane-bound NAC transcription factor SlNACMTF3 intomato plants under heat stress

Stress is perceived at the cellular membrane, where membrane-bound transcription factors (MTFs) are activated, initiating a rapid gene-regulation cascade. We previously demonstrated the multifaceted role of tomato MTF, SlNACMTF3, in regulating expression of biotic and abiotic stress responsive genes. However, the molecular mechanism underlying its activation following stress perception remained unknown. Here, we show that the membrane rhomboid protease SlRP3 recognizes an ISK motif near the transmembrane domain of SlNACMTF3 for cleavage and releasing the transcription factor from the membrane. SlRP3 and SlNACMTF3 co-localize within the same plasma membrane microdomain and interact directly. Molecular dynamics simulations supported the proposed mechanism, in which heat-induced conformational switching of SlRP3, driven by reorientation of the L5 loop, enhances substrate accessibility and proteolytic activity. Furthermore, mutation of key SlRP3 residues involved in substrate interaction impaired SlNACMTF3 activation and reduced the expression of stress33 responsive genes. Analysis with SlNACMTF3-overexpressing tomato lines and SlRP3 transient knockdown leaves further revealed that transcription factor activation, stress35 responsive gene regulation, and cellular ROS detoxification occur coordinately. Collectively, these findings provide mechanistic insight into stress-triggered SlNACMTF3 activation and identify SlRP3 as a novel membrane-associated stress sensor that activates a major transcriptional regulator to enhance stress preparedness in economically important tomato plants.

plant biology↗

bioRxiv2026

Modelling the human IL6RA Asp358Ala variant in mice: effects on atherosclerosis and abdominal aortic aneurysm

The association of IL6RA Asp358Ala variant with reduced risk of atherosclerotic cardiovascular disease (ASCVD) and abdominal aortic aneurysm (AAA) prompted interest in targeting IL6 signalling in these conditions. A recent report that ziltivekimab, a neutralising anti-IL6 antibody, did not reduce major adverse cardiovascular events in patients with ASCVD and chronic kidney disease, has cast doubt on the causal role of IL6 signalling in ASCVD. Here, we show that introducing the orthologous IL6RA Glu357Ala substitution in male mice leads to the same alteration in soluble IL6RA levels seen in humans, and significantly prevents atherosclerosis and AAA. Thus, therapies that more closely reproduce the biological effects of the IL6RA Asp358Ala variant on IL6 signalling are needed to reduce cardiovascular risk in humans.

pathology↗

bioRxiv2026

A Versatile Microfluidic Device for High-throughput Combinatorial Drug Screening

Combination therapies can improve anticancer efficacy, but identifying effective drug pairs and dose combinations requires systematic exploration of multidimensional concentration spaces. Here, we extend a diffusion-driven, flowless microfluidic concentration gradient generator (CGG) to enable quantitative combinatorial drug screening without external pumps or continuous flow. The platform comprises a 5 x 5 array of interconnected culture nodes coupled to four peripheral reservoirs, in which overlapping diffusion fields generate spatially defined single- and multidrug exposures. Computational modelling was used to assign local drug concentrations to individual nodes, enabling direct correlation of the predicted exposure landscape with cellular response. Using MCF-7 breast cancer cells and 5-fluorouracil (5-FU) and doxorubicin (DOX) as model therapeutics, the platform resolved concentration-dependent single-agent responses, yielding IC50 values of 3.46 M for 5-FU and 1.22 M for DOX. Combinatorial loading generated 25 spatially defined 5-FU-DOX concentration pairs within a single device, which were resolved into two-dimensional concentration-response landscapes. Bliss independence analysis revealed concentration-specific drug interactions, with synergy predominating at low-to-intermediate concentrations and a transition towards additive and antagonistic responses at higher exposures. These findings establish a pump-free microfluidic strategy that integrates computational concentration mapping with spatially resolved pharmacological analysis to identify effective drug-combination windows within a single platform.

cancer biology↗

bioRxiv2026

Target-Specific Discovery of BMM_1567 Restores Aminoglycoside Activity Against Multidrug-Resistant Gram-Negative ESKAPE Pathogens

The global emergence of multidrug-resistant (MDR) ESKAPE pathogens has significantly reduced the effectiveness of existing antibiotics, highlighting the urgent need for new strategies to restore antimicrobial susceptibility. Here, we report the discovery and mechanism of BMM_1567, a peptide potentiator that enhances aminoglycoside efficacy against MDR pathogens. A genetically defined reporter-based screen identified BMM_1567 as a potent inhibitor of aminoglycoside resistance, potentiating spectinomycin activity against MDR Gram-negative ESKAPE isolates at low micromolar concentrations. Structural modeling and molecular dynamics simulations indicated that BMM_1567 interacts with residues lining the antibiotic-binding groove of aminoglycoside-modifying enzymes (ANT, APH, AAC), with highest affinity for ANT ({Delta}G_bind = -62.25 kcal/mol), suggesting competitive inhibition of substrate binding. Site-directed mutagenesis of key ANT residues identified critical amino acids involved in BMM_1567 binding, confirming their role in mediating spectinomycin potentiation. In murine abscess model using XDR E. coli, BMM_1567 in combination with spectinomycin significantly reduced bacterial burden and pro-inflammatory cytokine levels, comparable to colistin. Collectively, these findings establish BMM_1567 as a promising aminoglycoside potentiator that restores antibiotic activity against MDR pathogens through direct inhibition of resistance enzymes, while exhibiting in vivo efficacy and a remarkably low propensity for resistance development.

microbiology↗

bioRxiv2026

Strand Displacement Activity of Mimiviral Polymerase X Enables Rapid Detection of Sequence-Specific DNA Targets

Mimiviral polymerase X, mvPolX, is a repair polymerase that is involved in base excision repair (BER) and carries out the gap-filling function in double-stranded DNA (dsDNA). We demonstrate a sensitive and sequence-specific DNA detection method using this polymerase. mvPolX begins polymerizing DNA from the 3 end of a gap, displacing the downstream nucleotides without exonuclease activity. Our detection method is built on this activity of mvPolX. We designed a probe molecule consisting of a partial dsDNA with a 3 over-hang region complementary to the target DNA to be detected. The probe has a fluorophore-quencher (FAM-BHQ1) tag to facilitate detection upon strand removal. Binding of the probe to the complementary target forms a dsDNA with a single nucleotide gap in one strand. mvPolX binds this gap region and begins polymerisation eventually displacing the quencher strand leading to an increase in fluorescence. Proof-of-concept has been established using a synthetic 19 bp target DNA sequence. The method is specific and did not show any strand displacement when a single or double mismatched nucleotide at the 3 end of the target DNA was used. To demonstrate this molecular assay, we used M13 phage as our target. Asymmetric PCR (aPCR) was used to obtain single-stranded target DNA (158 bases) from M13 genomic DNA, which was directly used in the assay as target. The combination of aPCR and mvPolX assay can detect as low as 10 copies of genomic DNA. The enzymatic reaction is fast, requiring only 15 min of incubation with mvPolX at 30 {degrees}C. We have further demonstrated the efficiency of the assay in presence of multiple non-target DNA by detecting the target DNA from M13 phage spiked lakewater samples.

molecular biology↗

bioRxiv2026

T-cell Receptor (TCR) targeting with Multivalent T-cell Engagers

T-cell engagers (TCEs) for cancer immunotherapy have traditionally relied on high affinity single chain fragment variable (scFv) domains to target CD3, specifically the {varepsilon} chain, for the activation of T-cells. Despite their clinical success, there have been reports of TCEs driving systemic toxicity, non-specific T-cell activation, on-target off-tumor effects, and severe inflammation due to cytokine release. To address these limitations, we designed multivalent TCEs using Chemically Self-Assembled Nanorings (CSANs) that target the /{beta} constant region of the T-cell receptor (TCR) in the TCR/CD3 complex using a moderate affinity TCR nanobody (TCRVHH). Nanobodies offer superior physical and chemical properties over scFvs-including higher solubility, stability and lower production cost-making them increasingly popular as structural units of TCEs. We compared the efficacy and safety profile of this moderate affinity, nanobody-based TCR binder against high affinity CD3scFv based CSANs across EGFR and PSMA expressing solid tumor models. While the CD3scFv CSANs offered potent cytotoxicity, they also induced antigen independent T-cell activation bypassing the requirement of tumor crosslinking for cytotoxicity. In contrast the TCRVHH CSANs required strict antigen engagement to trigger cytotoxicity, significantly reducing non-specific T-cell activation and thus enhancing the safety profile. Although the initiation of cytotoxicity was kinetically slower than the CD3scFv counterpart, TCRVHH CSANs achieved comparable end point cytotoxicity across multiple antigen densities, as well as in 3D tumor spheroids. Through this study we demonstrate the applicability of nanobodies as T-cell targeting domains, enhanced specificity and safety of moderate affinity T-cells binders and the diversification of T-cell targeting epitopes without compromising the efficacy of TCEs. Abstract Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/725710v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@9bcb83org.highwire.dtl.DTLVardef@4e30daorg.highwire.dtl.DTLVardef@1695931org.highwire.dtl.DTLVardef@15ec7ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Somatic mutations reveal the ontogeny of human microglia

Microglia are the resident hematopoietic cells of the central nervous system1. In mice, microglia seed the brain during embryogenesis and can be maintained throughout life with minimal input from adult hematopoiesis2-4. The origins of human microglia are less clear, but recent evidence suggests that marrow-derived cells may be able to supplement the human microglial pool in certain individuals5,6. Here, to investigate the ontogeny of human microglia, we develop a method that uses the collection of accumulated somatic mutations which uniquely labels each clone of cells to track the infiltration of marrow-derived cells into the human brain. Applying this method to 20 aged individuals, we find evidence of an influx of marrow-derived cells into the brain in all examined individuals. Single cell analysis, including single cell lineage tracing using mitochondrial DNA variants, demonstrates that these infiltrating cells are nearly identical to microglia and can comprise a large fraction of the microglial pool. Analysis of large-scale sequencing cohorts demonstrates a protective association between most types of clonal hematopoiesis and Alzheimers disease. In sum, this work uncovers a widespread influx of myeloid cells into the healthy human brain which serves to reinforce the pool of human microglia and becomes common with aging.

immunology↗

bioRxiv2026

Vegetation increases CH4 emissions and methanotroph diversity in marine sediments

Seagrass meadows are key blue carbon (C) ecosystems, storing large amounts of organic C over centuries. Their climate benefits may be reduced by methane (CH) emissions, whose microbial and environmental descriptors in Zostera noltei meadows, dominant seagrass in North-Western Europe, remain poorly understood. We studied CH fluxes, CH-producing and consuming microbial communities and sediment physico-chemical parameters in Z. noltei meadows and adjacent bare sediments across seven sites in Arcachon Bay, France. In situ CH fluxes were measured at low tide and microbial communities were characterised using targeted metagenomics of three functional genes (mcrA, mmoX, pmoA) and quantitative PCR. CH fluxes were higher in vegetated than bare sediments (24.4 {+/-} 2.6 vs. 9.4 {+/-} 0.7 {micro}mol m-{superscript 2} d-{superscript 1}). Mixed linear models and random forest analyses identified C accumulation rate and CO2 flux as the strongest positive descriptors of CH fluxes. Vegetated sediments hosted more diverse methanotrophs, while methanogens showed no habitat differences. Four genera (mcrA-Methanolobus, mmoX-Methylocella, pmoA-Methylococcus, Methyloglobulus) emerged as abundant, seagrass-associated, correlated with CH fluxes, and highlighted by models. Functional diversity, especially pmoA richness, was a stronger microbial descriptor of CH fluxes than gene abundance or a specific genus. Findings indicate Z. noltei meadows enhance C burial and CH emission, with methanotroph diversity potentially mitigating CH emissions. Our results provide the first integrated assessment of CH fluxes and their descriptors in Z. noltei meadows, highlighting the intertwined nature of C burial and CH emissions and the need to account for both in blue C climate assessments.

ecology↗

bioRxiv2025

Deep learning methods for automated bone segmentation and biomechanical modelling in zebrafish

Automated segmentation of three-dimensional micro-computed tomography (CT) scan data is a critical bottleneck in computational morphometrics and biomechanical modelling across musculoskeletal biology. Although advances in imaging have generated increasingly large and complex datasets, manual segmentation remains prohibitively time-consuming, while existing deep learning solutions are often application-specific and rarely validated for their impact on downstream analyses. Here we present a generalisable computational framework for automated segmentation and biomechanical validation of skeletal structures, implemented using an attention-augmented 3D U-Net architecture. Using the adult zebrafish (Danio rerio) mandible as a representative case study, the network was trained on 47 manually segmented specimens and evaluated using a combined Dice-Hausdorff metric that integrates both volumetric and surface accuracy to capture biologically relevant morphology better than Dice score alone. To assess performance in a downstream biomechanical context, we directly compared automated segmentations with those produced by three expert human annotators and constructed finite element models from each. Quantitative comparisons of segmentation accuracy and statistical analyses of finite element outputs demonstrate that automated segmentations perform within the range of expert human annotations, with no systematic bias in mechanical predictions. By explicitly linking geometric accuracy to biomechanical outcomes, this work establishes an end-to-end pipeline for validating automated segmentation in computational biology. The framework will be applicable to a wide range of musculoskeletal and palaeobiological contexts, including comparative anatomy, ageing and disease studies, and fossil or incomplete specimens, where scalable and mechanically faithful segmentation is essential. Author summaryMany studies now use CT scanning to build computer models of bones and test how they perform under load. However, these analyses usually start with manual segmentation, where an expert traces the bone in thousands of image slices to create a 3D surface. This step is time-consuming and introduces uncertainty because experts do not always agree exactly on where boundaries lie. If segmentation changes the model shape, it may also change predicted biomechanics. In this work, we test whether deep learning can automate segmentation while preserving biomechanical outcomes. We train an attention augmented 3D U-Net to segment the adult zebrafish lower jaw and compare its outputs directly to three expert manual segmentations. We assess similarity using complementary measures that reflect both overall overlap and boundary differences. We then build finite element models from each segmentation and compare predicted stress, strain, and displacement distributions. We show that automated segmentations are as consistent as human experts and generate indistinguishable biomechanical predictions, without systematic bias. This supports scalable, repeatable pipelines for linking anatomy to function, and provides a general approach for validating automated segmentation in studies where downstream modelling is the goal.

bioengineering↗

bioRxiv2025

LoAR-Exo and HiAR-Exo: One-step label-free isolation of extracellular vesicles using inertial microfluidic devices

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio [~] 1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 m from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

bioengineering↗

bioRxiv2025

dCas9 targeted proteome profiling reveals p300-mediated reciprocal regulation of SMAD and SP1 as a driver of GM2 synthase transcription in renal cell carcinoma

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasnt clear. This study demonstrated that the histone acetyl transferase (2), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

molecular biology↗

bioRxiv2025

PYM1 limits non-canonical Exon Junction Complex occupancy in a gene architecture dependent manner to tune mRNA expression

The Exon Junction Complex (EJC) deposited upstream of exon-exon junctions during pre-mRNA splicing in the nucleus remains stably bound to RNA to modulate mRNA fate at multiple post-transcriptional steps until its disassembly during translation. Here, we investigated two EJC disassembly mechanisms in human embryonic kidney 293 (HEK293) cells, one mediated by PYM1, a factor that can bind both the ribosome and the RBM8A/MAGOH heterodimer of the EJC core, and another by the elongating ribosome itself. We find that EJCs lacking PYM1 interaction show no defect in translation-dependent disassembly but is required for translation-independent EJC destabilization. Surprisingly, PYM1 interaction deficient EJCs are enriched on sites away from the canonical EJC binding position including on transcripts without introns or with fewer and longer exons. Acute reduction of PYM1 levels in HEK293 cells results in a modest inhibition of nonsense-mediated mRNA decay and stabilization of mRNAs that localize to endoplasmic reticulum associated TIS-granules and are characterized by fewer and longer exons. We confirmed the previously reported PYM1-flavivirus capsid protein interaction and found that human cells expressing the capsid protein or infected with flaviviruses show similar changes in gene expression as upon PYM1 depletion. Thus, PYM1 acts as an EJC specificity factor that is hijacked by flaviviruses to alter global EJC occupancy and reshape host cell mRNA regulation.

molecular biology↗

bioRxiv2024

Loss of FBXO31-mediated γH2AX foci formation impairs initiation of NHEJ and HR repair pathways, and sensitizes breast cancer to therapy

In response to genotoxic stress, cell initiates complex signalling cascades to combat genomic insults through simultaneous initiation of growth arrest and DNA damage repair process. {gamma}H2AX functions as a crucial initiator in DNA double strand damage repair process. Therefore, {gamma}H2AX foci formation onto the damage sites is essential to initiate the recruitment of repair proteins involved in NHEJ (Non-homologous DNA-end joining) or HR (Homologous recombination) repair process. However, molecular events associated with {gamma}H2AX foci formation onto the DNA damage sites are poorly understood. Here, we show that FBXO31, the first ubiquitin ligase, mediated Lys-63-linked ubiquitination of {gamma}H2AX is essential for its foci formation onto the DNA damage sites to initiate recruitment of proteins involved in NHEJ and HR-mediated DNA damage repair. Therefore, tumors with FBXO31 deficiency show enhanced growth suppression following chemotherapeutic drug treatment because of synthetic lethality, indicating that FBXO31 could be used as a marker for predicting the outcome of chemotherapy treatment.

cancer biology↗

bioRxiv2024

Evaluation of extraction solvents for untargeted metabolomics to decipher the DOM of Antarctic cryoconite holes

Cryoconite holes are biological hotspots with a high biogeochemical turnover rate, contributing significantly to the glacial ecosystems overall carbon cycles and net fluxes. Unfortunately, the information about the composition of low molecular weight molecules formed through the metabolic processes of cryoconite-dwelling microbes is scanty. These molecules constitute a substantial portion of the dissolved organic matter (DOM) within cryoconite holes. The present study investigated the composition of DOM in cryoconite holes using reverse-phase liquid chromatography (RP-LC) coupled with high-resolution tandem mass spectrometry. We evaluated various solvent combinations of water, methanol, and acetonitrile to extract chemically diverse polar and non-polar metabolites from the cryoconite holes. Among the single solvents, organic-rich MeOH: Water (70:30 v/v) and in parallel 2-single solvent combinations of MeOH: Water (70:30 v/v) and Acetonitrile: Methanol: Water (40:40:20 v/v) provided increased number and chemical diversity of extracted metabolites. Combining RP with the hydrophilic interaction liquid chromatography (HILIC) technique provided the highest number of unique metabolites. This dual-LC and ionization polarity combination increased the detection of metabolic features by 46.96% and 24.52% in single- and two-solvent combinations compared to RP alone. This study developed a simple untargeted metabolomics workflow that is highly sensitive and robust, detecting and potentially identifying a large number of chemically diverse molecules present in the DOM (extracellular) and microbes (intracellular) from the cryoconite holes environment. This method can better characterize DOMs chemical composition and, after integrating with other omics approaches, can be used to examine the link between metabolic pathways and microbial communities in global cryoconite holes or other similar ecosystems, revealing how these earthy systems and their microbial flora control carbon or nutrient storage or release in response to global climate change. Overall, the study presents a valuable methodology for studying the biogeochemistry of cryoconite holes.

microbiology↗

bioRxiv2024

Damaging mutations in LXRα uncouple lipogenesis from hepatotoxicity and implicate hepatic cholesterol sensing in human liver health

The nuclear receptor Liver X Receptor- (LXR) activates lipogenic gene expression in hepatocytes. Its inhibition has therefore been proposed as a strategy to treat metabolic-dysfunction-associated steatotic liver disease (MASLD). In order to understand the impact of reducing LXR activity on human health we first examined the association between the carriage of rare loss of function mutations in NR1H3 (encoding LXR) and metabolic and hepatic phenotypes. We identified 63 rare predicted damaging variants in the ligand binding domain of LXR in 454,787 participants in UK Biobank. On functional characterisation, 42 of these were found to be severely impaired. Consistent with loss of the lipogenic actions of LXR, carriers of damaging mutations in LXR had reduced serum triglycerides ({beta}=-0.13 s.d. {+/-}0.03, P=2.7x10-5, N(carriers)=971). Surprisingly, these carriers also had elevated concentrations of serum liver enzymes (e.g. ALT: {beta}=0.17s.d. {+/-}0.03, P=1.1x10-8, N(carriers)=972) with a 35% increased risk of clinically significant elevations in ALT (OR=1.32, 95%CI:1.15-1.53, P=1.2x10-4, N(carriers)=972), suggestive of hepatotoxicity. We generated a knock-in mouse carrying one of the most severely damaging mutations (Nr1h3 p.W441R) which we demonstrated to have dominant negative properties. Homozygous knock-in mice rapidly developed severe hepatitis and fibrotic liver injury following exposure to western diet despite markedly reduced steatosis, liver triglycerides and lipogenic gene expression. This phenotype was completely rescued by viral over-expression of wildtype LXR specifically in hepatocytes, indicating a cell-autonomous effect of the mutant on hepatocyte health. While homozygous LXR knockout mice showed some evidence of hepatocyte injury under similar dietary conditions, the phenotype of the LXRW441R/W441R mouse was much more severe, suggesting that dominant negative mutations that actively co-repress target genes can result in pathological impacts significantly more severe than those seen with simple absence of the receptor. In summary, our results show that loss of function mutations in LXR occur in at least 1/450 people and are associated with evidence of liver dysfunction. These findings implicate LXR in the maintenance of human liver health, identify a new murine model of rapidly progressive fibrotic liver disease and caution against LXR antagonism as a therapeutic strategy for MASLD.

genetics↗

bioRxiv2024

Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair complex

Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins sense damage within genomic DNA. We examined DNA dynamics related to damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Using laser temperature-jump (T-jump), we measured the dynamics of DNA containing 3 base-pair mismatches recognized specifically by Rad4 in vitro. The T-jump kinetics traces measured using a cytosine-analog FRET pair, together with rigorous comparison with equilibrium measurements, enabled conformational dynamics to be revealed beyond the T-jump observation window of [~]20 {micro}s - 50 ms. AT-rich nonspecific sites (matched or mismatched) exhibited dynamics primarily within the T-jump window, albeit with some amplitude in "missing" fast (< 20 {micro}s) kinetics. The fast-kinetics amplitudes increased dramatically for specific sites, which were further distinguished by additional missing amplitude in slow (> 50 ms) kinetics at elevated temperatures. We posit that the rapid ({micro}s-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >50-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that likely govern how Rad4 senses DNA damage.

biophysics↗

bioRxiv2023

Exploring the Role of MMP9 and its Interaction with TGFβ Signalling in Patients with Mesial Temporal Lobe Epilepsy-Hippocampal Sclerosis

Inflammation and blood brain barrier (BBB) damage are associated with epileptogenesis in Mesial Temporal lobe epilepsy with Hippocampal sclerosis (MTLE-HS). Animal studies have predicted the role of Matrix metalloproteinase 9 (MMP9) in extracellular matrix (ECM) modulation, BBB leakage and neuro-inflammation, while Transforming growth factor beta (TGF{beta}) signalling in astrocytes potentiates hyper-excitability leading to seizure generation. We hypothesize whether changes in activity and expression of MMP9, and the ratio of MMP9 and its inhibitor, Tissue inhibitor of metalloproteinase 1 (TIMP1), have a role in epileptogenesis in the patients with MTLE-HS through zona occludens 1 (ZO1) modulation. We also proposed the role of astrocytic TGF{beta} signalling in these patients. mRNA expression of MMP9 and TIMP1 was significantly up-regulated. The ratio of MMP9 to its inhibitor TIMP1 was greater than one, suggesting activation of MMP9, further confirmed by gelatin zymography. MMP9 activity as well as immunoreactivity was higher in patients with MTLE-HS as compared to non-seizure controls, whereas the immunoreactivity of ZO1 was significantly lower in the patients. The downstream TGF{beta} signalling effector molecules, SMAD3 and pSMAD3 immunoreactivity were also significantly higher in MTLE-HS patients and both molecules showed co-localisation with astrocytes in the hippocampal region. Further, we showed preliminary data about interaction of MMP9 and TGF{beta}1 in these patients as evidenced by a co-immunoprecipitation assay. This study highlighted the MMP9 and astrocytic TGF{beta} signalling mediated potential mechanism of epileptogenesis in MTLE-HS patients.

neuroscience↗

bioRxiv2023

Measuring RBC deformability and its heterogeneity using a fast microfluidic device

We report a high-throughput microfluidic device to determine the Youngs modulus of single red blood cells (RBCs). Our device consists of a single channel opening into a funnel, with a semi-circular obstacle placed at the mouth of the funnel. As a RBC passes the obstacle, it deflects from its original path. Using populations of artificially-stiffened RBCs, we show that the stiffer RBCs deflect more compared to the healthy RBCs. We then generate a calibration curve that maps each RBC trajectory to its Youngs modulus obtained using an atomic force microscope. Finally, we sort a mixed population of RBCs based on their deformability alone. Our device could potentially be further miniaturized to sort and obtain the elastic constants of nanoscale objects, such exosomes, whose shape change is difficult to monitor by optical microscopy.

bioengineering↗