Biology subjects

Paul, S.

Publications and source records attributed to Paul, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Stepwise Proteinase K Digestion Improves Genomic DNA Yield and Purity from Goat Semen: A Comparative Pilot Study of Five Extraction Protocols

Background: Genomic DNA of high quality is a prerequisite for molecular genetic analyses. However, DNA extraction from goat semen is frequently challenged by highly compacted sperm chromatin and the presence of enzymatic inhibitors within the seminal plasma. Objectives: This pilot study aimed to identify a reliable and cost-effective method for isolating high-quality genomic DNA from caprine semen. Methods: Five extraction protocols were evaluated: an unmodified blood protocol (Protocol B), two modifications of a sperm protocol (Modified Protocols 1 and 2), and a stool protocol (Protocol BS) using a commercial spin-column kit (Kit A), alongside a second commercial silica membrane kit (Kit B). Three technical replicates per protocol were processed from a single frozen semen straw of a Black Bengal buck (with one replicate evaluated for Protocol B). Extracted DNA was evaluated based on concentration, purity (absorbance ratios A260/A280 and A260/A230), cost per sample, and PCR amplification success of the mitochondrial hypervariable region 1 (HVR1). Results: Modified Protocol 2 utilizing 100 uL of semen and two sequential 1.5-hour proteinase K incubations at 55 degrees C yielded the highest normalized DNA concentration (mean +/- SD: 55.9 +/- 30.4 ng/uL semen), optimal purity ratios (A260/A280 = 1.78; A260/A230 = 2.06), and the lowest cost per nanogram of DNA (INR 0.073 +/- 0.037). All extracts from this protocol were successfully amplified via PCR. While the Kruskal-Wallis test for normalized yield did not reach statistical significance (p = 0.075), rendering yield differences exploratory, Modified Protocol 2 demonstrated the strongest overall performance. Conclusions: Modified Protocol 2 offers a promising, cost-efficient workflow for caprine sperm DNA isolation. Given that this pilot study sampled a single animal, these findings must be validated using biological replicates from multiple bucks prior to widespread implementation.

molecular biology↗

bioRxiv2026

Gut bacterium of Leishmania donovani-non-transmitting sand fly induces IL-23~Th17 axis to protect hosts from drug-resistant visceral leishmaniasis

Background: Visceral leishmaniasis (VL) is caused by Leishmania donovani and differentiates under the influence of biotope-specific gut bacteria in the Leishmania donovani-transmitting sand fly vector, Phlebotomus argentipes, which coexists with the Leishmania donovani-non-transmitting Sergentomyia species in the Indian subcontinent. Hypothesis: Sergentomyia are primarily herpetophilic. Interestingly, we identified a specific population of anthropophilic Sergentomyia babu babu, a proven naturally non-competent vector for transmitting Leishmania donovani, with high abundance and human-bite incidences in VL-non-endemic regions, compared with VL-endemic regions of India. Thus, the possibility of egesting gut bacteria to humans from VL-non-transmitting Sergentomyia and inducing downstream immune cascades remains undeniable. Innovation: We characterized gut bacteria from anthropophilic Sergentomyia babu babu in both VL-endemic and VL-non-endemic regions of India and found that a heat-inactivated formulation of Heyndrickxia sporothermodurans [NCBI Sequence Read Archive (SRA): PRJNA1403236], isolated from anthropophilic Sergentomyia babu babu from a VL-non-endemic region, prevented and cured L. donovani infection by inducing IL-23~Th17. More importantly, it also prevented the initial parasite establishment by inducing phagocytosis, ROS, and TNF- in circulatory Ly6G-CD11b+Ly6Chi monocytes. To the best of our knowledge, this is the first report confirming that the gut bacteria of VL-non-transmitting sand flies are critical and need to be investigated in depth for developing therapeutics and/or prophylactics against visceral leishmaniasis (VL), and suggests that possible bite-transfer of gut bacteria from disease-non-transmitting vectors to hosts may restrict disease pathogenicity. Further clinical trials are needed to warrant (Indian patent: 202511041013, April 28, 2025).

immunology↗

bioRxiv2026

DNA polymerase kappa supports repair-associated DNA synthesis and neuronal genome resilience

Postmitotic neurons must preserve genome integrity for decades despite continuous oxidative, transcription-associated, and environmental DNA damage. Although recent studies have established ongoing repair-associated DNA synthesis in mature neurons, the polymerases that execute this synthesis remain incompletely defined. DNA polymerase kappa (POLK), a Y-family translesion synthesis polymerase, is highly expressed in neurons and undergoes an age-associated redistribution from the nucleus to the cytoplasm, suggesting that loss of nuclear POLK may compromise neuronal genome maintenance. Here, we investigated the consequences of POLK loss and augmentation in primary mouse cortical neurons and human induced pluripotent stem cell-derived forebrain neurons, including APOE e4/e4 backgrounds. Partial POLK depletion reduced neuronal arbor complexity, enlarged neuronal nuclei, increased LAMIN A/C and nuclear-envelope abnormalities, and induced a multidimensional senescence-like stress phenotype characterized by increased senescence-associated {beta}-galactosidase, p21, nuclear p38 MAPK, inflammatory signaling, lysosomal remodeling, and reduced mitochondrial-associated staining. POLK localized in proximity to EdU-labeled DNA in mature neurons, and POLK depletion reproducibly reduced EdU incorporation across independent human neuronal backgrounds and maturation stages. Exploratory nuclear POLK immunoprecipitation-mass spectrometry, together with proximity ligation assays, placed POLK near proteins involved in nucleotide excision repair, base-excision/single-strand break repair, DNA-end repair, checkpoint signaling, and specialized polymerase regulation, including PCNA-Ub and RAD18. POLK loss also produced Alzheimer disease-relevant cellular phenotypes. In wild-type neurons, POLK depletion increased 6E10 immunoreactivity and was accompanied by remodeling of APP/endosomal, lipid/cholesterol, and lysosomal pathways. In APOE e4/e4 neurons, POLK depletion increased cytoplasmic dsDNA and phospho-tau. Conversely, neuron-directed POLK augmentation increased EdU incorporation while reducing 53BP1 and {gamma}H2AX signals, p38 MAPK, cytoplasmic dsDNA, 6E10 immunoreactivity, and phospho-tau. Together, these findings identify POLK as a component of repair-associated DNA synthesis in mature neurons and support a model in which neuronal POLK availability contributes to genome maintenance, stress resilience, and vulnerability in aging and Alzheimer disease-relevant states.

neuroscience↗

bioRxiv2026

Selective Hippocampal Reactivation Driven by Neocortical Feedback Supports Memory Transfer in Predictive-Coding Networks

During sleep and quiet wakefulness, the brain spontaneously revisits neural activity patterns associated with previous experience, a phenomenon known as reactivation, or replay. Reactivation is thought to support systems consolidation, the process by which memory retrieval becomes progressively more dependent on neocortical circuits and less dependent on the hippocampus. Importantly, reactivation is not indiscriminate: it preferentially targets specific memories, such as new or weakly learned ones. These observations have motivated the development of computational models accounting for the spontaneous reactivation of memories, their prioritisation for replay, and their transfer between the hippocampus and neocortex. In parallel, predictive-coding (PC) models have shown how computations attributed to hippocampal and neocortical circuits can arise from local prediction-error dynamics. Here, we connect these two lines of work by showing how autonomous intrinsic dynamics in coupled PC networks can transfer stored memory content between neural systems. The hippocampus and neocortex are modelled as two coupled PC associative memory networks, with asymmetric coupling motivated by reported interactions between hippocampal area CA1 and the prefrontal cortex. This coupling establishes a closed-loop dialogue in which hippocampal activity drives neocortical learning, while neocortical feedback biases subsequent hippocampal activity toward content for which neocortical support remains weak. We prove that this feedback steers hippocampal activity toward states of higher neocortical free energy, so that prioritisation emerges from the dynamics rather than from an explicit scheduler. In our model, neocortical learning relies exclusively on spontaneous hippocampal activity, without access to the initial training corpus. Nevertheless, we show that the neocortical network acquires correlated memory content faster than direct training on randomly sampled memories. This advantage appeared for sufficiently correlated memories, grew with their correlation, and persisted when new memories were incrementally added during transfer. Together, these results provide a PC account in which neocortical feedback organises hippocampal reactivation to support efficient memory transfer during systems consolidation.

neuroscience↗

bioRxiv2026

Plasma Extracellular Vesicle Lipidomics Reveals an SMPD1-Driven Sphingomyelin Salvage Pathway Collapse in Parkinson's Disease

While lysosomal lipid dysregulation is implicated in Parkinsons disease (PD), identifying the precise enzymatic drivers remains limited by the lack of subcellularly resolved metabolic mapping. Leveraging endosome-origin plasma extracellular vesicle (PsEV) lipidomics, we identified a profound bidirectional disruption of the ceramide-sphingomyelin (SM) axis in PD, driven by asymmetric upregulation of sphingomyelin phosphodiesterase 1 (SMPD1) and sphingomyelin synthase 1 (SGMS1). BODIPY-C5 SM tracking revealed that SMPD1 perturbation severely impairs lysosomal SM turnover. Crucially, this SMPD1-driven hydrolysis forces a salvage pathway collapse, bottlenecked by SPHK1/SGPL1 downregulation, which precipitates oxidative membrane damage and PINK1/Parkin-dependent mitophagy failure. We robustly validated this multi-pathway dysfunction across orthogonal in vitro, in vivo mouse, and postmortem human brain models. Furthermore, SMPD1/SGMS1 expression distinctly stratifies clinical phenotypes, marking the aggressive Postural Instability/Gait Difficulty (PIGD) subtype. Our findings establish SMPD1-mediated lysosomal impairment and ceramide-SM imbalance as a core biochemical axis in PD pathophysiology.

neuroscience↗

bioRxiv2026

Discovery of a Small-Molecule mTORC1 Pathway Activator that Enhances Adoptive T-Cell Therapy via Immuno-metabolic Reprogramming of CD8+ T Cells

Adoptive cell therapy (ACT) has transformed cancer immunotherapy; however, its clinical efficacy remains limited by progressive T cell dysfunction and inadequate metabolic fitness acquired during ex vivo expansion and within the tumor microenvironment. Here, we report the discovery of AS-3, a first-in-class small-molecule activator that enhances the therapeutic competence of CD8 T cells through RAPTOR-dependent mTORC1 activation. Identified by phenotypic screening of an in-house library of pharmacologically relevant scaffolds and subsequent medicinal chemistry optimization, AS-3 markedly increased effector cytokine production while preserving T cell viability. Mechanistically, AS-3 enhanced phosphorylation of mTORC1 downstream effectors, promoted glycolytic and mitochondrial metabolism, and sustained pathway activity under rapamycin-mediated inhibition. Transcriptomic profiling of activated human CD8 T cells revealed coordinated enrichment of mTORC1 signalling, oxidative phosphorylation, glycolysis, proliferative programs, and cytotoxic effector gene networks, consistent with comprehensive immunometabolic reprogramming. Genetic silencing of RAPTOR attenuated both mTORC1 signalling and cytokine induction, establishing pathway dependency. Ex vivo conditioning with AS-3 significantly improved antitumor efficacy in two independent adoptive T-cell therapy models, accompanied by enhanced persistence, reduced exhaustion, and superior effector function. Together, these findings establish pharmacological activation of the RAPTOR-mTORC1 axis as a strategy to generate metabolically resilient T cells and provide a clinically translatable approach to improving the durability and efficacy of next-generation ACT.

immunology↗

bioRxiv2026

Remodelling of chromatin architecture and super-enhancer landscape in lamin A/C depleted HGSOC

Lamins are nuclear intermediate filament proteins that maintain nuclear architecture through interactions with the chromatin. The A- and B-type lamins tether the genome at the peripheral lamina underlying the inner nuclear membrane in the form of heterochromatic lamina-associated domains (LADs). However, LADs associated with A-type lamins are confined not only to lamina but also to the nuclear core, thereby pointing to their distinct and multifarious roles in chromatin organisation and regulation. Previous studies involving lamin B1 depletion depicted the detachment of LADs from the nuclear periphery, accompanied by alteration of chromatin distribution while preserving the topologically associating domains (TADs) in structurally intact form. In this piece of work, we have shown, for the first time in HGSOC, the effects of lamin A/C knockdown on chromatin organisation, which was characterised by significant detethering of gene-poor chromatin from the periphery along with active (A) to inactive (B) compartment switching. This was associated with a rewiring of oncogenic super-enhancer elements corroborated by the differential gene expression profile. Overall analysis tipped the scale in favour of reduced cellular proliferation upon lamin A/C knockdown. This finding was strengthened by the observed proliferative potential of spheroids ex vivo and tumours in a mouse xenograft model.

cancer biology↗

bioRxiv2026

Understanding the physiological alterations of Vibrio cholerae upon exposure to L-ascorbic acid

The scourge of cholera remains a major global public health threat. It affects up to 4 million people worldwide and causes tens of thousands of deaths each year. The disease is experiencing a concerning resurgence in many parts of Africa, the Middle East, and Asia. To effectively tackle cholera and circumvent rising antimicrobial resistance, targeted biological and preventive approaches, complementing traditional rehydration, are urgently needed. In this regard, our group has demonstrated the efficacy of L-ascorbic acid in controlling the growth and pathogenesis of Vibrio cholerae in vitro. The present work further provides a mechanistic elucidation of the L-ascorbic acid-mediated physiological changes in V. cholerae and also bolsters such a non-antibiotic approach to control cholera.

microbiology↗

bioRxiv2026

Viral Protease Nsp5 Hijacks La Autoantigen to Orchestrate the Translation to Replication Transition in SARS-CoV-2

Viruses genetically encode proteins to regulate their life cycle. To translate this genetic information, most viruses exploit host translation machinery. Host dependency leads to competition between viral RNA and host mRNAs. Most viruses overcome it by inhibiting cap-dependent translation of host mRNAs and shifting its own translation into cap-independent mode. This switch is largely orchestrated by host RNA binding proteins (RBPs) by binding to viral 5UTRs. Here we demonstrate that human La autoantigen (La protein) specifically binds to GCAC sequence near the initiator AUG within the 5UTR of SARS-CoV-2 RNA promoting viral non-structural protein synthesis by cap independent translation, while suppressing replication early in infection. Interestingly, around 18-24 h post-infection, the viral protease Nsp5 cleaves La at a conserved LQ site, abrogating its 5'UTR interaction, which correlates with reduced polysome association and increased viral RNA availability for replication. MD simulation and docking study suggests that the cleavage disrupts La dimerization and truncates its C-terminal intrinsically disordered region, reducing RNA-binding affinity and conformational stability required for translation-competent ribonucleoprotein complexes. Results put forward a unique mechanism by which Nsp5-mediated proteolysis of La drives the temporal switch from translation to replication of SARS-CoV-2.

microbiology↗

bioRxiv2026

Preclinical Characterization of Hippo pathway inhibition: Insights from Pharmacological and Genetic Studies

The Hippo pathway is an evolutionarily conserved regulator of growth, regeneration, and organ homeostasis, and while its dysregulation is well established in cancer, the effects of inhibiting this pathway on normal tissues are less understood. Here we have systematically investigated the impact of Hippo pathway inhibition by comparing pharmacologic perturbation using a covalent small-molecule TEAD inhibitor (TEADi CMPD1, also known as GNE-8025) with genetic suppression of YAP/TAZ. We identified three key target organs that consistently emerged upon TEAD inhibition: the kidney, as well as the pancreas, and thymus. Across models, both perturbations led to comparable disease phenotypes in these organs, including tubular degeneration in the kidney, acinar atrophy in the pancreas, and lymphoid depletion in the thymus. However, the extent of damage was more pronounced in mice treated with the small-molecule inhibitor, highlighting potential dose and compound specific effects while remaining broadly consistent with the phenotypes observed upon genetic ablation of YAP/TAZ. This highlights the key role of evaluating both genetic and pharmacological perturbations to characterize the phenotypes and potential toxicities when modulating novel targets in oncology. To further investigate the mechanisms underlying pan-TEAD inhibition and kidney related adverse effects, we further characterized this class effect through a comprehensive transcriptomic analysis of the kidney to map the pathways involved in renal response. SignificanceUnderstanding on target toxicities is critical for the safe clinical development of TEAD inhibitors. Here, by integrating pharmacologic TEAD inhibition with genetic suppression by developing a mouse model that characterizes systemic, inducible knockdown of YAP/TAZ, we provide a systematic framework to define the Hippo pathway liabilities in vivo. We identify kidney, pancreas, and thymus as conserved target organs with concomitant phenotypes across both genetic and pharmacological methods, establishing these as pathway driven effects. Importantly, we uncover dose dependent and partially irreversible injury, particularly in kidney and pancreas, alongside mechanistic insight linking TEAD inhibition to aldosterone signaling disruption in kidney. These findings highlight the importance of strategies to identify monitorable, manageable adverse effect to guide clinical translation of TEAD targeting strategies.

cancer biology↗

bioRxiv2026

An Integrated Proteomics and Genomics Approach to Identify Essential Protein Kinases During Human Trophoblast Development

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrets human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) to profile proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1 and TNIK are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKC{zeta} is essential for STB development, while PTK2B, SRC, TRIO and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

developmental biology↗

bioRxiv2026

Legacy Effects of Land-Use Land-Cover Change on Species Distribution Dynamics: Evidence from a landscape of diverse biogeographic crossroads

Land-use and land-cover (LULC) change is a dominant driver of biodiversity loss, yet its long-term role in restructuring species distribution remains poorly understood due to limited historical baselines and the scarcity of long-term analyses across multiple ecological transition zones. Here, we investigate nearly two centuries of land-use change across a biogeographically diverse region to evaluate how landscape transformation has reshaped vertebrate distribution range dynamics, and whether ecoregional context, habitat specialization, and taxonomic identity better explain these patterns than recent landscape change alone. Using Texas, a biogeographic crossroads with diverse ecoregional zones and a long history of intensive land-use, we quantify distributional changes in 100 native vertebrate species (mammals, birds, reptiles, and amphibians), using Vernon Baileys late nineteenth-century surveys as a historical benchmark. We integrated multi-temporal LULC datasets with spatial and multivariate analyses to assess how habitat change, species traits, and regional context relate to patterns of range expansion, contraction, and redistribution. We show that Texas has undergone non-linear landscape transformations characterized by early agricultural expansion followed by cropland abandonment, shrubland encroachment, and rapid urbanization. Range contractions were most pronounced among amphibians, reptiles, and habitat specialists, whereas generalist species and many birds exhibited greater range stability or expansion. Across taxa, primary habitat, vertebrate class, and ecoregional context emerged as the strongest predictors of distributional change, underscoring the importance of historical and environmental context over simple measures of habitat loss and fragmentation. These results indicate that while long-term LULC change establishes the underlying landscape template, species responses are structured by ecological context, including habitat affinity, taxonomic traits, and regional environmental gradients. By linking historical landscape trajectories to contemporary biodiversity patterns, this study provides a transferable framework for investigating how land-use legacies influence species distributions and community reassembly in heterogeneous human-dominated landscapes, highlighting the importance of adaptive capacity and connectivity for conservation planning.

ecology↗

bioRxiv2026

Spatial Regulation of CAR Signaling Enables Logic-Gated Activity

Chimeric antigen receptors (CARs) can induce T cells to kill cancer cells but also to kill normal cells that express the same antigens1. Designing CARs to recognize combinations of antigens, via Boolean logic, can simultaneously expand the scope of targetable antigens and make CAR T cells more specific to cancer2. For example, one antigen may be expressed on cancer cells and normal bone marrow cells, while a second antigen may be present on the same cancer cells but only in normal lungs. If recognition of both antigens is required for T cell activation, only the cancer cells will be killed. Creating such AND-gated CAR T cells has been challenging given the need to engineer non-natural signaling mechanisms that integrate two ligand binding events into a single T cell activation stimulus3-6. Here, we design a fundamentally new AND-gated receptor called Multi-ANtigen Triggered Immune Synapse (MANTIS), which leverages differences in extracellular receptor dimensions to regulate CAR signaling. MANTIS initially prevents CAR activity by steric blocking with a bulky extracellular domain. Upon engagement of the first antigen, MANTIS sheds this blocking domain, releasing a free CAR that can bind a second antigen and activate the T cell in an AND-gated manner. This work demonstrates how differences in extracellular receptor size can be leveraged to spatially regulate intracellular signaling pathways in response to antigen patterns, paving the way for new applications in synthetic biology and cell engineering. One sentence summaryDifferences in extracellular size can be leveraged to regulate CAR T cell activity for precise recognition of antigen patterns.

synthetic biology↗

bioRxiv2026

The Georgetown Reading in Aging Neuroimaging Dataset (GRAND): Reading and multimodal MRI data in older adults

Reading is a critical skill in modern society. Most research on reading is conducted in school age children or young adults. However, acquired brain disorders often affect reading ability, and these disorders tend to occur in older adults. It is therefore critical to examine the normative distribution of reading behavior and the brain basis of reading in older adults. Here, we provide trial-wise single word and pseudoword oral reading and lexical decision data, as well as structural, functional, and diffusion-weighted MRI data from 116 neurotypical adults aged 22 to 84 years (mean = 59). Accuracy, response times, and errors are provided for corpora that are parametrically modulated in frequency, imageability, and regularity for real words and consistency of spelling-sound mapping for pseudowords. This dataset includes both minimally processed behavior (trial-wise data) and MRI data, and participant- and item-wise summary metrics and processed MRI data. These data serve both as a normative sample for reading behavior in older adults, but also as a valuable resource for identifying novel brain-behavioral relationships.

neuroscience↗

bioRxiv2026

Landscape-scale grassland structural heterogeneity and disturbance regimes influence swamp deer habitat utilisation across the upper Gangetic plains, India

Large herbivores play a vital role in shaping grassland ecosystems, yet many species face increasing threats from habitat loss, fragmentation, and human disturbance. In northern India, riverine grasslands of the Gangetic floodplains serve as essential habitats for such species, including the swamp deer (Rucervus duvaucelii duvaucelii), a habitat specialist that has experienced severe population declines, especially outside protected areas. We evaluated the structural vegetation composition of grasslands and swamp deer habitat use along the Ganges river, covering both protected regions (Jhilmil Jheel Conservation Reserve and Hastinapur Wildlife Sanctuary) and surrounding non-protected grasslands. Combining field-based vegetation surveys with NDVI-based remote sensing enabled the classification of grasslands into four ecologically significant types structured by Saccharum, Phragmites, and Typha species. Disturbance was prevalent across the landscape, with approximately 77% of grassland plots exhibiting signs of human impact. Pure Saccharum grasslands exhibited the highest disturbance levels, followed by mixed patches, while Phragmites-Typha patches showed the lowest disturbance. Conversely, Phragmites-Typha patches experienced the lowest levels of disturbance. Data from surveys and two radio-collared swamp deer indicated a consistent preference for mixed vegetation types, particularly Saccharum-dominated and Phragmites-Typha mixes, while avoiding heavily disturbed pure Saccharum patches. Seasonal changes in habitat use reflected context-dependent selection influenced by vegetation structure, hydrology, and disturbance regimes. Our results underscore the importance of conserving diverse grassland mosaics and reducing human pressures, especially in unprotected areas to promote the long-term survival of swamp deer and other grassland herbivores within the Gangetic floodplains.

ecology↗

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

Myeloid Gi signaling acts as a weight-independent immunometabolic switch controlling systemic insulin sensitivity

Metabolic dysfunction does not necessarily correlate with adiposity. Metabolically healthy obese individuals and insulin-resistant lean individuals represent a fundamental paradox that implicates immune cell intrinsic mechanisms in the pathogenesis of type 2 diabetes. Here, we identify myeloid Gi signaling as a previously unrecognized determinant of whole-body glucose homeostasis. Single-cell transcriptomic analysis of adipose tissue macrophages from obese mice and humans reveals marked alteration in Gnai isoform, suggesting that myeloid Gi signaling is functionally engaged during metabolic disease. Using complementary myeloid-specific rodent models of Gi inhibition (pertussis toxin) and chemogenetic Gi activation (DREADD), we demonstrate that inhibition of Gi signaling improves glucose tolerance and enhances insulin sensitivity under both regular chow and high-fat diet conditions, independent of body weight and energy expenditure. Whereas acute Gi activation in lean mice modestly enhances glucose disposal, the same intervention during diet-induced obesity markedly impairs systemic glucose homeostasis, revealing context-dependent pathway function. Mechanistically, Gi inhibition amplifies macrophage cAMP-CREB signaling to drive IL-6 production, engaging STAT3- and AMPK-dependent pathways in adipose tissue and skeletal muscle to support insulin action. Conversely, Gi activation engages a previously uncharacterized G{beta}{gamma}-mTOR/AKT-JNK cascade, driving IL-1{beta} secretion that directly impairs insulin signaling in adipocytes and myotubes. Pharmacological IL-6 receptor blockade abolishes the metabolic benefits of Gi inhibition, whereas IL-1 receptor antagonism fully rescues Gi activation-induced metabolic dysfunction, establishing these cytokines as obligate downstream effectors. This signaling architecture is conserved in human macrophages, and ATAC-seq profiling reveals chromatin remodeling at cAMP-CREB and IL-6 regulatory pathway loci, consistent with the observed transcriptional reprogramming. Together, these findings establish myeloid Gi signaling as a weight-independent immunometabolic switch that couples opposing cytokine programs to systemic insulin sensitivity and identify this pathway as a therapeutic target in obesity-associated metabolic disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/713834v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e4576org.highwire.dtl.DTLVardef@224cd5org.highwire.dtl.DTLVardef@1e24888org.highwire.dtl.DTLVardef@1080bf3_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

physiology↗

bioRxiv2026

Conserved RNA-protein modules link early anthracycline responses to atrial fibrillation risk

Anthracycline chemotherapeutics increase risk for cardiovascular disease. The early molecular events that link inherited risk to cardiac dysfunction remain poorly defined. We therefore exposed iPSC-derived cardiomyocytes from six individuals to three anthracyclines and an anthracenedione, which act as topoisomerase inhibitors (TOP2i) to induce DNA damage, and generated proteomics data following three and 24 hours of TOP2i treatment. We constructed a 19 module co-expression network and identified four protein modules that associate with response to all TOP2i. Integration with a co-expression network generated from paired transcriptomic data revealed that the two most drug-responsive protein modules are preserved in the RNA network. The most preserved RNA module is enriched for p53 motifs in associated active regulatory regions and p53 target genes, which propagates to enrichment of p53 targets in the most drug-responsive preserved protein module. Integration of the protein network with genome-wide association studies across hundreds of cardiovascular traits identified a preserved protein module enriched for genetic risk of atrial fibrillation, PR interval, and longitudinal strain, suggesting that the molecular response to TOP2i is linked to genetic variation influencing cardiac electrophysiology and contractile performance. Differential protein abundance in this module associates with impaired calcium handling in cardiomyocytes thereby linking molecular effects to cellular decline. Together, these results define a TOP2i-induced gene regulatory response propagated to the proteome that underlies early cardiotoxicity, and demonstrate how a multi-level network architecture can provide insight into genetically mediated susceptibility to drug-induced stress.

genomics↗