Biology subjects

Das, S.

Publications and source records attributed to Das, S..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Mapping of residues in leishmanial glyceraldehyde-3-phosphate dehydrogenase crucial for binding with 3'-UTR of TNF-alpha mRNA

Recently, we described that glyceraldehyde-3-phosphate dehydrogenase from Leishmania major (LmGAPDH) was present in extracellular vesicles and it inhibited host TNF- expression during infection via post-transcriptional repression. The LmGAPDH binding with the AU-rich elements in 3-untranslated region of TNF- mRNA (TNF- ARE) is sufficient for limiting this cytokine production, but the TNF- ARE binding residues in LmGAPDH are still unexplored. RNA electrophoretic mobility shift assay (REMSA) and catalytic activity measurement revealed that the inhibition by TNF- ARE was competitive with respect to cofactor NAD+ in LmGAPDH. To identify the TNF- ARE binding residues of the LmGAPDH, we exploited a systematic mutational analysis of its NAD+ binding domain. Catalytic activity measurement indicates that both R13 and N336 amino acids in the NAD+ binding site are absolutely required for activity whereas other mutants including I14A, R16A, D39A and T112A showed higher Km (lower affinity) value for NAD+ binding and lower catalytic activity. REMSA studies revealed that the replacement of Arg-13 with Ala/Lys or Asn-336 with Ala resulted in complete loss of binding with the TNF- ARE. I14A, R16A, D39A and T112A residues at or near NAD+ binding site showed lower binding with the TNF- ARE compared to the wild-type protein. The protein induced fluorescence enhancement (PIFE) studies and in vitro protein translation assay further confirmed the REMSA results. Based on our findings, the NAD+ binding residues in LmGAPDH are important for TNF ARE binding.

biochemistry↗

bioRxiv2025

Single-Cell Multimodal Profiling Highlights Persistent Aortic Smooth Muscle Cell Changes in Diabetic Mice Despite Glycemic Control

BackgroundType 2 diabetes (T2D) is associated with accelerated vascular complications like hypertension and atherosclerosis. "Phenotypic switching" of vascular smooth muscle cells (SMC), a major driver of these complications, is enhanced in diabetes. Despite adequate glycemic control, SMC dysfunction can persist due to "metabolic memory" of prior hyperglycemia. However, the mechanisms of hyperglycemic memory associated with persistent SMC dysfunction are unclear. Here, leveraging single-cell (sc) multi-omics, we examined the effect of glucose normalization on transcriptomic and epigenomic changes associated with SMC phenotypic transition in T2D mice. MethodsWe treated T2D db/db mice with the antidiabetic drug dapagliflozin (DAPA) (db/dbDAPA) or vehicle (db/db), and non-diabetic control db/+ mice with vehicle for 6 weeks. Dissected aortas were subjected to scRNA-seq, scATAC-seq, and spatial transcriptomics (Xenium) to determine single-cell changes in gene expression and chromatin accessibility. ResultsDAPA treatment conferred effective glycemic control in db/db mice, with significant reductions in blood glucose/hemoglobin A1c. scRNA and scATAC-seq analysis of aortas identified major cell populations, including SMC, fibroblasts, endothelial and immune cells. SMC were further clustered into 9 subtypes, including contractile and fibromyocyte-like cells. Cell composition analysis revealed decreases in contractile SMC and increases in vascular remodeling associated fibromyocyte-like SMC in db/db versus db/+ mice. Interestingly, DAPA did not reverse diabetes-induced decreases in contractile markers but reversed changes in several fibromyocyte markers in db/db mice. Pseudotime trajectory analysis revealed increased activities of fibromyocyte enriched transcription factors (TFs) during contractile to fibromyocyte transition. Furthermore, increased expression of TFs regulating fibromyocyte phenotype (e.g. Atf4, Bach1, Hand2, Fosl2) in db/db were partially reversed by DAPA, whereas reduced contractile TF (Mef2c) expression was unchanged. Spatial transcriptomics analysis further mapped aortic cell types within intact aortas and confirmed that DAPA reversed alterations in key fibromyocyte but not contractile genes in db/db mouse aortas. ConclusionsPersistent epigenetic changes may contribute to sustained vascular remodeling and dysfunction in T2D. T2D reduced contractile SMC gene expression and related chromatin accessibility and promoted phenotypic transition to fibromyocytes. These changes are only partially reversed by a widely used antidiabetic drug like DAPA, underscoring the need for more effective therapies that target hyperglycemic memory.

biochemistry↗

bioRxiv2025

Dysregulation of neuronal activity-dependent immediate early genes in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder triggered by impaired function of the maternal UBE3A gene, which codes a protein that functions as a ubiquitin ligase and transcriptional coactivator. Ube3a maternal deficient mice replicate many key behavioral deficits associated with AS; however, the underlying molecular mechanisms remain poorly understood. Using an RT2 Profiler PCR array that summarizes the expression of 84 genes regulating synaptic plasticity, we identified a number of dysregulated genes in the visual cortex of AS mice brains at postnatal day 25 (P25) compared to wild-type animals. In-depth analysis revealed that various immediate early genes (IEGs), like Arc, Egr1-4, and Homer, are dramatically down-regulated in the visual cortex of AS mice with regard to wild-type controls at P25. Moreover, the dark rearing of wild-type mice considerably decreased the levels of these IEGs to nearly the same level as those found in AS mice, along with the down-regulation of Ube3a. Furthermore, a significant reduction in the expression of these IEGs can also be observed in the hippocampus of AS mice. Finally, we demonstrate that the augmented activity of HDAC2 in the AS mice brain might be connected with the down-regulation of various IEGs and other synaptic plasticity-regulating genes. These findings indicate that the deregulated expression of neural activity-dependent IEGs could be linked with abnormal synaptic plasticity and associated behavioral deficits observed in AS mice.

neuroscience↗

bioRxiv2025

The Ventral Attention Network Mediates Attentional Reorienting to Cross-Modal Expectancy Violations: Evidence from EEG and fMRI

Our daily interactions with the world are shaped by sensory expectations informed by context and prior experiences, which in turn influence how we allocate our attention. Prominent predictive coding models suggest that sensory expectancy and attention interact but disagree on the precise mechanisms. One possibility is that the Ventral Attention Network (VAN), may play a role by facilitating attentional reorienting when expectancy is violated. To test this, we employed an auditory-visual trial-by-trial cueing paradigm in three experiments integrating EEG and fMRI to investigate the VANs role in violations of cross-modal expectancy. Behavioral results showed faster responses to expected targets, confirming the efficacy of cue-induced expectations in orienting attention to the expected target modality. EEG analyses revealed differences in early ([~]100 ms latency) event-related potentials (ERPs) to both auditory and visual stimuli when expectations were violated. Unexpected stimuli elicited significantly larger early-latency negative ERPs, across both modalities. Source localization of these ERPs and subsequent fMRI evidence revealed activation in the right VAN. Functional connectivity analyses further showed greater coupling between VAN regions and sensory cortices, with modality-specific pathways involving superior temporal gyrus (STG) for auditory and fusiform gyrus (FG) for visual targets. These findings demonstrate that expectancy violations recruit the VAN to reorient attention and resolve sensory conflict. By coordinating top-down control and bottom-up sensory input, the VAN supports adaptive responses to unexpected stimuli. This work advances our understanding of predictive processing in multisensory perception and highlights the VANs central role in flexible cognitive control.

neuroscience↗

bioRxiv2025

GPCR-targeted imaging and manipulation of homeostatic microglia in living systems

Microglia, the resident innate immune cells of the brain, are known to perform key roles such as synaptic pruning, apoptotic debris removal, and pathogen defense in the central nervous system. Microglial mutations are directly linked to many neurodevelopmental (e.g., schizophrenia) and neurodegenerative (e.g., Alzheimers disease) disorders, indicating the diagnostic and therapeutic potential of microglia for treating these conditions. Currently, we lack robust molecular tools to specifically image and manipulate microglia in vivo, which presents a major hurdle in our understanding of the brain-wide functions of these cells during the early onset of brain diseases. Here, we describe a molecular technology for imaging and manipulation of homeostatic microglia in live organisms (e.g., in zebrafish and mice) by covalently targeting the purinergic receptor, P2RY12. Using this technology, we imaged microglia-pathogen interactions in the larval zebrafish brain and revealed various morphological states of microglia in the adult mouse brain. We further expanded the microglia labelling approach to single-microglia tracking and microglial surfaceome mapping using photoactivatable fluorophores and photoproximity labelling, respectively. We anticipate the use of this universal tool for studying microglial biology across species to reveal the dynamics and polarization of resting microglia into a reactive state found in many neurodegenerative diseases.

immunology↗

bioRxiv2025

In vivo efficacy of L-ascorbic acid in restricting cholera pathogenesis

In the last three centuries, our world has experienced seven cholera pandemics. Cholera is a deadly diarrheal disease caused by Vibrio cholerae, an important member of the gamma-proteobacteria. Despite conventional treatments like antibiotics, vaccines, and ORS, the 7th cholera pandemic is still a major threat to developing nations. L-ascorbic acid was recently shown to effectively kill the Vibrio cholerae cells in vitro under various growth conditions mimicking the in vivo host conditions, including growth in the presence of bile salts, growth of acid-adapted V. cholerae, and growth in the presence of various ORS components. In the current study, we extend this work and test the efficacy of L-ascorbic acid in animal models (rabbit ileal loop and the removable intestinal tie adult rabbit diarrhea). We show that L-ascorbic acid can effectively reduce the bacterial load in both the rabbit models as well as fast-track the recovery from the diarrheal symptoms. ImportanceCholera is a severe human diarrheal disease that affects millions each year. Cholera treatment is primarily oral rehydration to balance the voluminous fluid loss. Previous studies have found that L-ascorbic acid (L-AA) is effective at inhibiting growth of Vibrio cholerae, the causative agent of cholera. Thus L-AA has potential as am inexpensive cholera therapeutic. Here, L-AA is tested in rabbit models to determine whether it can inhibit the effects of V. cholerae infection. Results suggest that L-AA treatment inhibits bacterial proliferation and leads to shortened recovery from disease.

microbiology↗

bioRxiv2025

mcDETECT: Decoding 3D Spatial Synaptic Transcriptomes with Subcellular-Resolution Spatial Transcriptomics

Spatial transcriptomics (ST) is a powerful tool for studying the molecular basis of brain diseases. However, most current analyses focus only on nuclear or somatic expression, overlooking distally localized mRNAs, known as the "dark transcriptome." These transcripts, which make up nearly 40% of the brain transcriptome, are packaged into RNA granules and transported to polarized neuronal structures critical for neuronal function. Here, we present mcDETECT, a machine learning framework that leverages in situ ST (iST) data to detect RNA granules and resolve their RNA composition. Applying mcDETECT to simulated and real datasets from multiple iST platforms, we demonstrate robust granule detection and molecular subtyping. Granule-informed downstream analysis further revealed functionally distinct neuronal substates not captured by somatic expression alone. In an Alzheimers disease (AD) mouse model, mcDETECT uncovered alterations in RNA granule distributions and associated neuronal substates prior to measurable neuronal loss, highlighting potential therapeutic targets for early AD pathology.

bioinformatics↗

bioRxiv2025

LuxS/AI-2 signaling in Salmonella Typhimurium regulates phoP/phoQ to protect against acidic pH for enhanced survival in macrophages

The intestinal milieu is largely characterized by the complex array of chemical compounds produced through the metabolic activity of resident microbiota. Enteric pathogens like Salmonella, which have evolved refined mechanisms to persist within this environment, utilize these microbial metabolites and self-produce quorum molecules as molecular cues to identify ecological niches and modulate their survival and virulence strategies. Salmonella quorum sensing involves producing and detecting Autoinducer-2 (AI-2) signaling molecules. Our research reveals that Salmonella Typhimurium enhances AI-2 biosynthesis and transport under acidic conditions, aiding environmental adaptation and facilitating pathogenesis in macrophages. AI-2 signaling regulates the pH-sensing two-component system genes, phoP/phoQ, ensuring cytosolic pH homeostasis, survival, and acid tolerance. It also involves regulating the lysine/cadaverine-mediated acid tolerance response and maintaining bacterial cytosolic pH. Furthermore, we show that the repressor LsrR protein, apart from the lsr promoter, binds to only one strand of the phoP promoter via its Y25 and R43 amino acids to negatively modulate phoP expression. Additionally this signaling ameliorates the intracellular survival by modulating Salmonella Pathogenicity Island-2 (SPI-2) regulators (ssrA/ssrB) and SPI-2 effector expression via PhoP. Mouse models demonstrate that AI-2 signaling is essential for colonizing the primary and secondary infection sites, showcasing its critical role in pathogenesis and low pH survival mechanisms in Salmonella.

microbiology↗

bioRxiv2025

Magnetized Cellbots to Spatiotemporally Control Differentiation of Human-Induced Pluripotent Stem Cells

Precise spatiotemporal control of gene expression and cellular differentiation is essential for engineering native-like multicellular structures. Current cell differentiation approaches typically rely on externally provided inputs whose effects are not targeted to distinct cells in the appropriate state and hence cannot spatially organize and mature tissue structures as needed. Our work introduces a magnetically controlled microrobot (MR) platform for guiding mammalian cells to desired locations that, combined with synthetic biology, delivers biological signals at precise locations and times, enabling spatiotemporal control of cell-fate decision-making. We use synNotch, a cell-cell contact-based biological signaling that induces relevant gene expression in receivers when the receiver cells contact sender cells through ligand-receptor binding. Magnetically driven MRs are then allowed to be internalized by sender cells, resulting in magnetized sender cellbots. Using a 3-pair orthogonal Helmholtz coil system, we guided magnetized sender cellbots to precise locations in a receiver cell culture, activating desired fluorescent protein expression in target Chinese Hamster Ovary (CHO) receiver cells. Next, we engineered Human-Induced Pluripotent Stem Cells (hiPSC) to function as receivers that can be instructed by senders to differentiate into endothelial cells (ECs) via overexpression of ETV2 (ETS variant transcription factor 2), a master transcriptional regulator of endothelial cell development. Using our magnetic platform, we guided multiple sender cellbots to target locations on a monolayer of hiPSC receivers, resulting in differentiation of receivers into ECs and possible onset of vascular formation. Our approach provides a foundation for the engineering spatial patterns by activating conditional triggers based on MR location and cell state at multiple time points, enabling several applications such as control of organoid architecture.

synthetic biology↗

bioRxiv2025

Comparative multi-omics analysis uncovers contrasting molecular profiles of canine and human thyroid carcinomas

Thyroid tumors represent 1-3% of canine cancers, with most tumors classified as follicular carcinomas and less frequently as medullary carcinomas. In comparison, only 10-15% of human thyroid cancers are follicular and 2% are medullary, with prevalent activating mutations in BRAF and NRAS, or RET, respectively. A cohort of canine thyroid carcinomas underwent histopathological (n=60 and paired molecular (n=30, WES and/or RNAseq) exploration. Clustering of tumor transcriptomes produced 2 groups; T1 and T2 clusters comprised of follicular thyroid carcinomas (FTC) and medullary thyroid carcinomas (MTC), respectively. Tumors were histologically typed as follicular, compact, and follicular-compact on blinded review, with most MTC classified as compact with rare follicular-compact appearance, while FTC displayed all 3 patterns. FTC samples had significantly elevated levels of ERBB2 and HER2 protein, and RET signaling was up-regulated in MTC. Recurrent somatic mutations in DNMT1, STAT2, SALL4, HSP90AA1, MEN1, MUC4, THRAP3, CDK4, NOTCH2, and THRAP3 were identified in at least 10% of samples. Individual variants were also identified in KRAS, ARAF, GNAS, and ERBB2. Additionally, we identified fusion genes that included TG, FGFR2, and PAX8. These data suggest that canine MTC, like their human counterparts, may be driven by RET signaling. In contrast, FTC show limited reliance on RAS/RAF signaling, prevalent in human TC, for oncogenic progression. Across the analyzed samples, 60% of tumors had mutations in at least one DNA repair-related pathway, suggesting that the accumulation of DNA damage may drive cancer progression in canine thyroid tumors. Elevated HER2 protein staining was associated with shorter progression free survival (PFS). Thyroid tumor size (>4.25 cm) was also associated with shorter overall survival and PFS, consistent with previous reports; however, metastasis at diagnosis was not correlated with outcome. Additional studies are warranted to explore the utility of these biomarkers to improve diagnosis and treatment of thyroid carcinoma in dogs.

cancer biology↗

bioRxiv2025

CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

The transcription-associated cyclin dependent kinases (CDKs) 12 and 13 play important roles in the expression of RNAs involved in DNA damage repair and ribosomal proteins, yet their roles if any, in the regulation of ribosomal DNA expression are unclear. Here we show that these CDKs play critical roles in the processing and degradation of noncoding RNAs in the nucleolus and that inhibition of their function leads to the upregulation of unprocessed ribosomal RNA (rRNA) transcripts in the intergenic spacer (IGS) regions between ribosomal subunits. These upregulated RNAs are aberrantly polyadenylated by the TRAMP complex polymerase TENT4, and are represented as polyadenylated rings surrounding the nucleolus. The accumulation of these noncoding RNAs is associated with increased RNA polymerase II (Pol II)-mediated transcription coupled with failure of degradation by the MTREX (SKIV2L2) RNA helicase, a member of the RNA exosome complex whose expression is prematurely terminated by CDK12/13 inhibition. Altogether, these findings provide new insights into Pol II-mediated transcriptional regulation by CDK12/13 within the nucleolus.

cancer biology↗

bioRxiv2025

Isolation and Characterization of Extracellular Vesicles and Non-Vesicular Extracellular Particles from Mouse Tissues

Extracellular vesicles (EVs) and non-vesicular extracellular particles (NVEPs) have been recently discovered as part of the diverse components of the secretome. EVs and NVEPs have been shown to differ not only in size and morphology but also in their cargo and biological functions. NVEPs are a newly discovered group of multimolecular assemblies with potential roles in physiological and pathological states. However, very little is known about this new class of particles. Here, for the first time, we present a method for simultaneously isolating and characterizing EVs and NVEPs from primary mouse tissues.

cell biology↗

bioRxiv2025

Predictive Coding Explains Asymmetric Connectivity in the Brain: A Neural Network Study

Seminal frameworks of predictive coding propose a hierarchy of generative modules, each attempting to infer the neural representation of the module one level below; the predictions are carried by top-down feedback projections, while the predictive error is propagated by reciprocal forward pathways. Such symmetric feedback connections support visual processing of noisy stimuli in computational models. However, neurophysiological studies have yielded evidence of asymmetric cortical feedback connections. We investigated the contribution of neural feedback during sensorimotor processes, in particular visual processing during grasp planning, by utilizing convolutional neural network models that had been augmented with predictive feedback and were trained to compute grasp positions for real-world objects. After establishing an ameliorative effect of symmetric feedback on grasp detection performance when evaluated on noisy stimuli, we characterized the performance effects of asymmetric feedback, similar to that observed in the cortex. Specifically, we tested model variants extended with short-, medium- and long-range feedback connections (i) originating at the same source layer or (ii) terminating at the same target layer. We found that the performance-enhancing effect of predictive coding under adverse conditions was optimal for medium-range asymmetric feedback. Moreover, this effect was most prominent when medium-range feedback originated at a level of representational abstraction that was proximal to the input layer, in contrast to more distal layers. To conclude, our simulations show that introducing biologically realistic asymmetric predictive feedback improves model robustness to noisy visual stimuli in a neural network model optimized for grasp detection. Significance statementIt is commonly held that the brain predicts the causes of its sensorium via top-down neural pathways. While canonical models of predictive coding assume reciprocal feedforward and feedback connections, functional evidence highlights the importance of non-reciprocal asymmetric feedback, whose role remains poorly understood, particularly in sensorimotor functions. Using neural network models of grasp planning, we characterized optimal pathlengths and source regions for asymmetric feedback facilitating visuomotor processing of noisy sensory inputs. Our findings show that medium-range feedback from early layers marks a sweet spot, incorporating optimal distance between the neural representations of source/target layers and representational abstraction of the feedback source. This intimates an uncharted role of intermediate brain areas along the visuomotor stream as a source of predictive signals.

neuroscience↗

bioRxiv2025

PI(3)P signaling regulates endosomal flux underlying developmental synaptic remodeling via Rab4

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes. Author summaryHealthy brain function relies on the growth and remodeling of synaptic connections between nerve cells. This process involves carefully orchestrated movements of proteins and membranes inside the neurites. For instance, endosome movement in the nerve cell extensions helps guide neuronal growth, recycle parts of the synapse, and alter signal transmissions between neurons. Rab GTPases, a group of small G-proteins, are crucial for these processes. Here, we aimed to understand what triggers these processes and how the supply chain within nerve cell extensions is connected to changes at the synapse. In fruit fly larvae, we observed that the movement of Rab4 vesicles in the nerve cell extensions is inversely related to gross synapse content. A further examination revealed that onset of insulin signaling accelerates the Rab4 vesicles towards synapse through a lipid kinase called Vps34, which makes a signaling lipid called phospatidylinnositol-3-phosphate. Higher levels this lipid molecule on Rab4 vesicles attract a motor protein, Klp98A, speeding up their movement during the synaptic contraction phase. Our findings reveal a new mechanism that regulates Rab4 vesicle transport and its impact on synapse stability. The study further suggests potential targets for controlling age-related dementia likely to be caused by excessive insulin signaling in neurons.

neuroscience↗

bioRxiv2025

Impact of Soil Salinity on Microbial Community Composition in Coastal Agricultural lands of Bangladesh: A Metagenomic Approach

A significant obstacle to agricultural productivity is soil salinity, especially in coastal areas where the problem is made worse by climate change and rising sea levels. This study investigates the impact of salinity on microbial community structure and function in agricultural soils from Kuakata, Bangladesh, using 16S rRNA metagenomic sequencing technique. Both saline (EC > 4 dS/m) and non-saline (EC < 1.5 dS/m) areas had soil samples taken; saline soils showed symptoms of crop stress, such as yellowing and blackish discoloration. In comparison to their non-saline counterparts, our investigation showed that saline soils had much reduced microbial diversity and different community compositions. Both soil types were dominated by Proteobacteria and Chloroflexi, while saline soils displayed an enrichment of anaerobic texa (e.g., Desulfobacterota, Pseudomonas) and sulfate-reducing bacteria (e.g., Desulfobacterota, Deferrisomatota). Interestingly, saline soils had a much larger percentage of anaerobic microorganisms (36.4%) than non-saline soils (29.3%) which might results in enhanced denitrification, sulphate reduction, and hydrogen sulphide buildup. These processes disrupt nutrient cycling, lower organic matter decomposition and create toxic conditions for plant growth, ultimately contributing to crop failure. This study provides an insight into the distinct microbial composition in saline and non-saline samples, which can assist in understanding the role of microbial populations in agriculture in saline environments.

microbiology↗

bioRxiv2025

Dynamical interactions among compositionally distinct protocell populations and its implications for evolution of early membranes

The spontaneous self-assembly of single chain amphiphiles (SCAs) would have resulted in multiple protocell species in an early-Earth niche. Considering the heterogeneity inherent in a prebiotic milieu, interactions between physicochemically distinct protocell populations was evaluated to discern if emergent properties occurred at a systems level. This study demonstrates that depending on the physicochemical properties of the membrane, interacting populations are endowed with varied emergent properties owing to their coexistence. In a multispecies paradigm involving a two-candidate protocell system, the fitter population acted as a predator and grew at the expense of the less-fit prey population. The observed growth could be attributed to the predator attaining a more robust membrane via chemical evolution. Importantly, the prey population also accrued emergent properties like molecular crowding, and coexist in balance with the predator population, without being completely outcompeted. When extrapolating these results to a three-candidate population, the outcomes were multipronged. These findings suggest a possible route for protocell membrane evolution that could have occurred even in the absence of any sophisticated protein machinery, benefitting coexisting populations, while also illustrating evolutionary trajectories that potentially resulted in functionally complex protocells.

biophysics↗

bioRxiv2025

Global transcriptome analysis identifies nicotinamide metabolism to play key roles in IFN-gamma and nitric oxide modulated responses

Interferon-gamma (IFN-{gamma}) is a key regulator of immune responses. A hallmark of the IFN-{gamma} response is inducible nitric oxide (NO) production, driven primarily by nitric oxide synthase 2 (NOS2). In this study, we investigated the influence of NO on the IFN-{gamma}-induced transcriptomic and metabolic changes in the RAW 264.7 macrophage cell line. IFN-{gamma} activation led to NO-dependent lactate production and lower cell survival. Bulk RNA sequencing analysis identified genes differentially expressed early by IFN-{gamma} that were either NO-independent or NO-dependent. Inhibition of NO modulated a minor subset of the transcriptome, notably affecting Klf6 (a tumor suppressor) and Zfp36 (a regulator of pro-inflammatory cytokines). Interestingly, both Klf6 and Zfp36 correlatively showed high expression in most cancers. The PPI network exhibited dense clustering with scale-free and small-world properties, identifying Stat1, Irf7, Irf1, Cxcl10, and Isg15 as top five hubs. The top IFN-{gamma} signalling genes exhibited deficient expression in the brain but were highly expressed in lung, spleen, and EBV-transformed lymphocytes. Gene-disease associations linked the IFN-{gamma}-regulated genes to immunodeficiencies, chronic inflammatory responses and malignancies. Interestingly, IFN-{gamma} upregulated genes were involved in nicotinamide metabolism, suggesting a transcriptional basis for modulation of metabolic pathways. This novel aspect was experimentally tested to show that IFN-{gamma} induced NAD amounts. Importantly, the inhibition of purine nucleoside phosphorylase (using Forodesine hydrochloride) which is involved in the endogenous pathway for NAD generation, lowered IFN-{gamma} induced nitrite and increased cell survival in vitro. Functionally, enriched nicotinamide metabolism by IFN-{gamma} may regulate inflammatory responses and the implications of our findings are discussed.

immunology↗

bioRxiv2025

Development of conditional-siRNA programmable riboswitch for targeting adverse cardiac remodeling

Heart Failure (HF) remains a global epidemic and a significant healthcare burden, with an unmet need for novel therapies to target the preceding pathological hypertrophy in vulnerable patients. Here we report the development of novel conditional-siRNA (Cond- siRNA) constructs that are selectively activated by disease-specific RNA biomarkers to enable cell-specific inhibition of a target disease-causing RNA. We designed a Cond- siRNA that can be activated by nppa mRNA, upregulated specifically in CMs under pathological stress, to silence the key pro-hypertrophic gene calcineurin by the effector siRNA. In cellular models including neonatal rat ventricular myocyte (NRVM) and rat cardiomyocyte cell-line (H9C2), Cond-siRNA exhibited low baseline activity in the absence of the disease biomarker but achieved targeted calcineurin silencing upon nppa mRNA induction by phenylephrine (PE)-induced stress in a two-dimensional (2D) cell culture system and pressure overload in three-dimensional (3D) heart-on a chip system. NRVM transfection with the Cond-siRNA resulted in a decreased expression of calcineurin mRNA specifically after PE or pressure-overload treatment, but not after vehicle treatment, proving nppa mRNA-specific activation of the effector siRNA against calcineurin. Specificity was confirmed as Cond-siRNA did not significantly silence calcineurin in cardiac fibroblasts and T cells, lacking nppa expression. Reduced calcineurin protein levels and NFATc1 nuclear translocation correlated with decreased NRVM hypertrophy after PE treatment, confirming Cond-siRNAs efficacy. This study offers proof-of-concept for Cond-siRNA as a targeted therapy to mitigate hypertrophic progression, paving the way for novel HF treatments. One sentence summaryConditional-siRNA targeting adverse cardiac remodeling GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/633434v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1b6532borg.highwire.dtl.DTLVardef@10e3836org.highwire.dtl.DTLVardef@8c839eorg.highwire.dtl.DTLVardef@1152883_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗