Biology subjects

Sharma, A.

Publications and source records attributed to Sharma, A..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Using phylogenetic relationships to assess conservation priorities for birds in India

In the midst of the ongoing biodiversity crisis, setting conservation priorities has moved beyond the conventional approaches. The need to incorporate phylogeny-based metrics that capture evolutionary rarity with extinction risk for prioritization of global conservation efforts has been widely recognized. Such metrics are even more important in megadiverse countries of the Global South, where conservation spending is limited despite high conservation value. Here, we reassess global avian threatened evolutionary history with an updated phylogeny, using the EDGE metric. The EDGE metric or score combines evolutionary distinctiveness of a species with its extinction risk. We focus on India to identify species and regions that represent high avian EDGE scores. We find that the Bengal Florican (Houbaropsis bengalensis) had the highest median EDGE score of 18.83 MY (millions of years). To identify spatial conservation priorities, we mapped avian EDGE scores at a 10 km x 10 km scale. Northeastern India is exceptional in its richness of evolutionary unique and threatened species. We also scored IBAs and PAs, finding that IBA, as well as PA, with the maximum EDGE score, was Intaki or Ntangki National Park in Nagaland, accumulating [~]250 MY of threatened evolutionary history in India. Habitats of the top 5% of EDGE species in India are currently not adequately protected. Our study offers a preliminary basis for a quantitatively informed and phylogeny-driven conservation prioritization approach for researchers, practitioners, and policymakers to safeguard threatened evolutionary history of birds in India.

evolutionary biology↗

bioRxiv2025

Ectopic expression of the sensor CovS cytosolic domain confers phosphatase activity and full virulence to the M1T1 CovSY39H attenuated variant of group A Streptococcus

Group A Streptococcus (GAS) causes a wide variety of diseases ranging from mild, noninvasive, such as pharyngitis and impetigo, to life-threatening infections, such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The two-component CovR/S system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence. An attenuated pharyngeal colonizing variant (S126) possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H) was recovered in France from a member of a family in which another individual developed NF and STSS caused by the M1T1 WT strain (S119). We employed transcriptome analyses (RNA-seq), quantitative determinations of CovR phosphorylation, measurements of virulence factor activity, and a murine model of human NF to demonstrate that CovS of strain S126 almost lost its entire phosphatase activity but retained its kinase and phosphotransfer activities. Moreover, we reversed its attenuated phenotype by ectopically expressing the cytosolic domain of wild-type CovS. Culturing the corresponding strain S126covS-3 in a chemically defined medium (CDM) supplemented with asparagine (Asn), conditions that produce an excess of cytosolic ADP over ATP, stimulated the ectopically expressed phosphatase activity. Consequently, this led to dephosphorylation of CovR[~]P and increased the expression of virulence factors. Most importantly, S126covS-3 reverted to the wild-type phenotype of S119 in the mouse model of human GAS NF. Our study provides a new mechanistic tool that enables the manipulation of CovS phosphatase activity both in vitro and in vivo. IMPORTANCEThe shift from high to low virulence and back in GAS is critical for understanding its pathogenesis and developing new treatments to control GAS infections. The two-component CovR/S system, comprising the sensor kinase/phosphatase CovS and the transcription regulator CovR, regulates the degree of GAS virulence. In the M1T1 serotype, covR/S mutations are typically associated with a loss of CovR/S function, leading to hypervirulent phenotypes. However, an attenuated pharyngeal-colonizing variant possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H strain S126) was isolated in France. Here, we demonstrate that S126 is attenuated because the mutation in CovS inhibits its phosphatase activity but preserves its kinase and phosphotransfer activities. By expressing the cytosolic domain of WT CovS in the S126 background, we endowed the resulting strain, S126covS-3, with phosphatase activity, which was further stimulated by ADP when formed in chemically defined medium (CDM) supplemented with asparagine (Asn). Most importantly, S126covS-3 switched to full virulence, similar to that of S119 in a mouse model of human GAS NF. These findings underscore the importance of comprehensive analyses of disease-related covR/S mutants in understanding the virulence and persistence of GAS.

microbiology↗

bioRxiv2025

The Old genetically heterogeneous Mouse Model Recapitulates Chronic and Persistent Idiopathic Pulmonary Fibrosis with Strong Senescence Signatures

Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive lung disease that primarily afflicts people over the age of 65. IPF is characterized by lung scarring, an elevated senescence burden, and interstitial pneumonia, resulting in disability and mortality. The growing aging population worldwide, limited effectiveness of current treatments, and high economic burden underscore the need for robust models to investigate the underlying mechanisms and test novel interventions. Despite broad preclinical use, the bleomycin-induced murine model has notable limitations. Animals subjected to a single dose of bleomycin administration undergo recovery in weight and behavior between 21 and 28 days after administration, which is contrary to the progressive nature of IPF. Previous reports have shown that repetitive instillation of bleomycin phenocopies this aspect of the human disease. However, these methods are time-consuming and complex. Although IPF is typically associated with advanced age, most research is conducted in 6 to 8-week-old mice, which lack the age-related structural and metabolic deficits seen in humans. In this study, we report an improved model of IPF using 17-month-old UM-HET3 mice subjected to a single oropharyngeal bleomycin dosing that better mimics the persistent nature of the disease. Lung histology and immunohistochemistry (IHC) confirm persistence of fibrosis and senescence in mice 10 weeks after bleomycin administration. Furthermore, bulk RNA sequencing (RNA-Seq) analysis revealed a distinct set of gene expression signatures that is more consistent with chronic human fibrosis. This model offers greater insight into IPF pathogenesis, and we anticipate that it will enhance confidence in the human translatability of candidate therapeutic interventions.

molecular biology↗

bioRxiv2025

Chemogenetic and optogenetic strategies for spatiotemporal control of split-enzyme-based calcium recording

Methods for monitoring physiological changes in cellular Ca2+ levels have been in high demand for their utility in monitoring neuronal signaling. Recently, we introduced SCANR (Split-Tobacco Etch Virus (TEV) protease Calcium-regulated Neuron Recorder), which reports on Ca2+ changes in cells through the binding of calmodulin and M13 to reconstitute an active TEV protease. First-generation SCANR marked all of the Ca2+ spikes that occur throughout the lifetime of the cell, but it did not have a mechanism for controlling the time window in which recording of physiological changes in Ca2+ occurred. Here, we explore both chemical and light-based strategies for controlling the time and place in which Ca2+ recording occurs. We describe the adaptation of six popular chemo- and opto-genetics methods for controlling protein activity and subcellular localization to the SCANR system. We report two successful strategies, one that leverages the LOV-J optogenetics system for sterically controlling protein interactions and another that employs chemogenetic manipulation of subcellular protein distribution using the FKBP/FRB rapamycin binding pair.

neuroscience↗

bioRxiv2025

Oxidative Stress, Hypoxia and Cellular Metabolism: Unraveling the Effects of Fentanyl on Lung Cancer Cells

Fentanyl, a widely used opioid analgesic for cancer pain management, is effective but requires cautious administration due to its potential for respiratory depression. Beyond its analgesic properties, fentanyls broader impact on cancer biology and biochemical alterations in lung carcinoma cells remains underexplored. This study investigates fentanyls influence on oxidative stress, mitochondrial function, hypoxia-inducing factors, apoptosis, and cytokine production in A549 lung cancer cells. Our findings reveal that fentanyl increases reactive oxygen species (ROS) generation, disrupts cellular homeostasis, induces DNA damage, and alters key signaling pathways, potentially affecting tumor metabolism and progression. Our "Ramanomics" data further highlight fentanyl-driven changes in key ions (inorganic phosphate, calcium) and biomolecules (glycogen, phospholipids, proteins, nucleic acids, and enzymes) at subcellular mitochondrial levels. These insights contribute to understanding fentanyls mechanistic impact on lung cancer progression and may inform optimized therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/665412v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1fb7aaaorg.highwire.dtl.DTLVardef@13255deorg.highwire.dtl.DTLVardef@1a398cdorg.highwire.dtl.DTLVardef@184d63e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Examining the composition of gut microbiota in a South African population: a comparative study between type 2 diabetes mellitus patients and non-diabetic individuals

BackgroundLiterature has highlighted the gut microbiotas role in metabolic functions, suggesting a potential link between gut microbiota composition and T2DM. The purpose of the study was to identify microbial signatures unique to T2DM patients and non-diabetic individuals, to compare microbial profiles between the two groups and to investigate how gut microbiota may be related to inflammation associated with T2DM. MethodsA cross-sectional study was conducted involving 51 T2DM patients and 99 non-diabetic South African individuals. Faecal samples were collected and analysed using 16S rRNA gene sequencing to characterize the gut microbiota. Blood samples were obtained to perform HbA1c, CRP and ferritin tests. Bioinformatic and statistical analyses were performed to identify differences in microbial composition and diversity between the two groups. ResultsThe gut microbiota in T2DM patients was predominantly composed of Firmicutes (47.7%), Bacteroidota (37.5%), and Proteobacteria (11.4%), while the non-diabetic group showed a slightly different microbial profile with higher Bacteroidota (41.9%) and a notable presence of Actinobacteriota (4.5%). Abundant families in the T2DM group included Bacteroidaceae (22.8%), Prevotellaceae (7.4%), Enterobacteriaceae (7.4%), Erysipelotrichaceae (6.0%) and Lachnospiraceae (5.2%). The non-diabetic group exhibited dominant families such as Lachnospiraceae 26.7%, Prevotellaceae (25.3%), Bacteroidaceae (12.7%), Ruminococcaceae (9.5%) and Oscillospiraceae (3.8%). At the genus level, Bacteroides (22.8%), Escherichia-Shigella (5.0%), Holdemanella (4.8%), Phascolarctobacterium (3.2%) and Blautia (2.8%) were prevalent in the T2DM group, while Prevotella_9 (22.1%), Bacteroides (12.7%), Agathobacter (6.7%), Blautia (6.3%) and Faecalibacterium (5.1%) were dominant in the non-diabetic group. Differential abundance testing revealed 5 phyla, 16 families, and 25 genera that were either enriched/depleted in T2DM patients relative to non-diabetic individuals. The comparison of alpha diversity metrics between the two groups revealed significant differences across all four measures (P < 0.001), with non-diabetic individuals showing higher values than T2DM patients. HbA1c and CRP levels showed correlations with the relative abundance of various gut microbes at various phyla, family, and genus levels, as well as with all alpha diversity metrics. ConclusionThe study revealed distinct differences in gut microbiota composition between T2DM patients and non-diabetic individuals, with T2DM patients showing a higher prevalence of certain phyla, families, and genera linked to metabolic dysregulation. Non-diabetic individuals exhibited greater microbial diversity and beneficial taxa, highlighting a potential protective microbial profile.

microbiology↗

bioRxiv2025

A Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP) for Extracellular Electron Transfer

Electroactive microbes can be used as components in electrical devices to leverage their unique behavior for biotechnology, but they remain challenging to engineer because the bioelectrochemical systems (BES) used for characterization are low-throughput. To overcome this challenge, we describe the development of the Bioelectrochemical Crossbar Architecture Screening Platform (BiCASP), which allows for samples to be arrayed and characterized in individually addressable microwells. This device reliably reports on the current generated by electroactive bacteria on the minute time scale, decreasing the time for data acquisition by several orders of magnitude compared to conventional BES. Also, this device increased the throughput of screening engineered biological components in cells, quickly identifying mutants of the membrane protein wire MtrA in Shewanella oneidensis that retain the ability to support extracellular electron transfer (EET). BiCASP is expected to enable the design of new components for bioelectronics by supporting directed evolution of electroactive proteins. The bigger pictureDevices that interface microbes and materials, known as bioelectronics, can be used to sense environmental chemicals in real time, generate energy from sugars, and synthesize chemicals. While these devices leverage the unique capabilities of living systems as components in devices, such as their ability to convert chemical information in the environment into electrical information at the cell surface, it remains challenging to engineer these cellular components and their biomolecules for new applications, largely because commercially available bioelectrochemical systems for monitoring current generated by electroactive microbes are costly and require large culture volumes, needs continuous monitoring for days to obtain stable signals, and multichannel potentiostats to monitor multiple microbes in parallel. To overcome these challenges, we created the Bioelectrochemical Crossbar Architecture Screening Platform or BiCASP that is easy to fabricate, enables parallel analysis of microbial samples in flexible arrayed formats, and yields a stable signal on the minute time scale. This device is expected to enable the application of combinatorial protein engineering methods, such as directed evolution, to proteins that control microbial current production, by allowing for fast screening of cells expressing protein mutant libraries. As a proof-of-concept, we demonstrate that this device can screen for cells that express mutants of decaheme cytochromes that retain the ability to electrically connect cells to electrodes. This device will simplify the engineering of cells and proteins that function as electrical switches as well as the diversification of bioelectronic devices for real-time sensing of chemicals in the environment. Furthermore, BiCASP is promising as a high-throughput screening (HTS) platform, enabling rapid, parallel analysis of cellular and molecular interactions of diverse biological systems through label-free electrochemical methods. Such capabilities could transform drug discovery, personalized medicine, and functional genomics, supporting systematic genetic and chemical screens even at single-cell resolution. HighlightsO_LIA high-throughput screening platform with individual addressability C_LIO_LIA device with a flexible crossbar architecture that simplifies current analysis C_LIO_LIReproducible detection of real-time cellular current on the minute time scale C_LIO_LIThe device can be used to screen a library for cells with functional protein wires C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=187 HEIGHT=200 SRC="FIGDIR/small/663982v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@c451d1org.highwire.dtl.DTLVardef@d7a50corg.highwire.dtl.DTLVardef@194e986org.highwire.dtl.DTLVardef@16f7af9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

scHDeepInsight: A Hierarchical Deep Learning Framework for Precise Immune Cell Annotation in Single-Cell RNA-seq Data

Immune cell classification from single-cell RNA sequencing (scRNA-seq) presents significant challenges due to complex hierarchical relationships among cell types. We introduce scHDeepInsight, a deep learning framework that extends our previous scDeepInsight model by integrating a biologically-informed classification architecture with an adaptive hierarchical focal loss. The framework leverages our established method of transforming gene expression data into two-dimensional structured images for use with convolutional neural networks by effectively capturing both global and fine-grained transcriptomic features, overcoming the limitations of flat classification approaches that ignore hierarchical relationships between cell types. scHDeepInsight dynamically adjusts loss contributions to balance performance across the hierarchy levels. It also employs STACAS batch correction, robust random masking, and SHAP-based interpretability to enhance prediction accuracy and biological insight. Comprehensive benchmarking across seven diverse tissue datasets shows scHDeepInsight achieves an average accuracy of 93.2%, representing a 5.1 percentage point improvement over current state-of-the-art methods. The model successfully distinguishes 50 distinct immune cell subtypes with high accuracy, demonstrating proficiency for identifying rare and closely related cell subtypes. These advantages make scHDeepInsight a robust tool for high-resolution immune cell subtype characterization, well suited for detailed immune profiling in immunological studies.

bioinformatics↗

bioRxiv2025

An M13 phagemid toolbox for engineering tuneable DNA communication in bacterial consortia

Intercellular communication is essential for distributed genetic circuits operating across cells in multicellular consortia. While diverse signalling molecules have been employed--ranging from quorum sensing signals, secondary metabolites, and pheromones to peptides, and nucleic acids--phage-packaged DNA offers a highly programmable method for communicating information between cells. Here, we present a library of five M13 phagemid variants with distinct replication origins, including those based on the Standard European Vector Architecture (SEVA) family, designed to tune the growth and secretion dynamics of sender strains. We systematically characterize how intracellular phagemid copy number varies with cellular growth physiology and how this, in turn, affects phage secretion rates. In co-cultures, these dynamics influence resource competition and modulate communication outcomes between sender and receiver cells. Leveraging the intercellular CRISPR interference (i-CRISPRi) system, we quantify phagemid transfer frequencies and identify rapid-transfer variants that enable efficient, low-burden communication. The phagemid toolbox developed here expands the repertoire of available phagemids for DNA-payload delivery applications and for implementing intercellular communication in multicellular circuits.

synthetic biology↗

bioRxiv2025

Emetine dihydrochloride inhibits Chikungunya virus nsP2 helicase and shows antiviral activity in the cell culture and mouse model of virus infection

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes frequent epidemics of chikungunya fever across the world and has become a significant medical challenge, warranting the development of novel antivirals. A cell imaging-based high-content screening of the Spectrum collection of small molecules, containing all the US and international drug molecules, identified Emetine dihydrochloride (ED) as a potent antiviral against CHIKV. ED inhibited CHIKV replication in the C57BL/6 mouse model of chikungunya disease, resulting in significantly lower viremia. Importantly, clinical symptoms of joint swelling were altogether absent in the CHIKV-infected mice treated with ED. In the ERMS cells, ED inhibited the CHIKV uptake, and the virus replication early during the virus life cycle by inhibiting the viral RNA synthesis, resulting in the inhibition of the viral protein synthesis. ED was predicted to bind the helicase domain of the CHIKV non-structural protein nsP2 by in silico methods. The ED binding to the nsP2 protein and its helicase domain was validated in vitro by microscale thermophoresis and isothermal titration calorimetry. Importantly, ED inhibited the CHIKV nsP2 helicase activity in vitro in a dose-dependent manner. These data demonstrate the potential of ED to be repurposed as a novel CHIKV antiviral, warranting clinical development. AUTHOR SUMMARYChikungunya virus (CHIKV) spreads through the bites of the virus-infected Aedes mosquitoes. The virus causes frequent epidemics of chikungunya fever involving severe joint edema and muscle pain. The virus activity has been recorded in more than 100 countries in Asia, Africa, South and Central America, and Europe, and it has become endemic in several regions of the world. In 2024, around 480,000 CHIKV cases and over 200 deaths were reported worldwide. While a CHIKV vaccine was recently approved, no virus-specific antiviral therapy is available. Considering the global medical significance of the virus, the development of effective CHIKV antivirals is a priority. With a view to repurposing an existing drug, we screened a collection of all the approved US and international drug molecules and identified Emetine dihydrochloride (ED) as a potent inhibitor of CHIKV replication in the cell culture model. The CHIKV-infected mice treated with ED showed reduced virus replication and no joint swelling, which is a typical clinical symptom of CHIKV disease. Our studies show that ED binds the CHIKV nsP2, inhibiting its helicase action, which is required for replicating the viral genomic RNA and producing the viral proteins. In the past, emetine has been used in humans to treat amoebiasis. Our work thus shows the potential of the drug emetine to be repurposed for treating CHIKV patients.

microbiology↗

bioRxiv2025

SP1 amplifies the effects of cellular proliferation via the Wnt/β-catenin pathway in colon development and cancer

Wnt/{beta}-catenin signaling is a highly conserved pathway across multicellular organisms, playing a pivotal role in various cellular processes essential for development and tissue homeostasis. Aberrant activation of this pathway is a major driver of colorectal cancer (CRC). The transcription factor SP1 has been identified as a key component of the Wnt/{beta}-catenin signaling pathway through its physical interaction with {beta}-catenin in colorectal cancer. However, the distinct roles played by this complex during development versus in disease contexts remain poorly understood. In this study, we conducted molecular and functional analyses by individually depleting SP1 and {beta}-catenin. Interestingly, genes co-regulated by the SP1:{beta}-catenin complex were significantly enriched for functions related to the cell cycle and DNA replication--key processes required for sustaining proliferation, whose disruption can promote tumorigenesis. These findings were further supported by data from mouse tumor models and clinical datasets. Additionally, cross-species analysis using zebrafish larvae revealed a synergistic interaction between SP1 and {beta}-catenin, leading to excessive cell proliferation and developmental defects. Collectively, our results suggest a conserved role for the SP1:{beta}-catenin complex in regulating cell proliferation during development and maintaining cellular homeostasis, processes critical for preventing cancer progression.

cancer biology↗

bioRxiv2025

Acid ceramidase inhibition enhances BCL-2 targeting in venetoclax-resistant acute myeloid leukemia via a cytotoxic integrated stress response

Resistance to combination regimens containing the BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) is a growing clinical challenge for this extensively utilized agent. We previously established the anti-leukemic properties of ceramide, a tumor-suppressive sphingolipid, in AML and demonstrated that upregulated expression of acid ceramidase (AC), a ceramide-neutralizing enzyme, supported leukemic survival and resistance to BH3 mimetics. Here, we report the anti-leukemic efficacy and mechanisms of co-targeting AC and BCL-2 in venetoclax-resistant AML. Analysis of the BeatAML dataset revealed a positive relationship between increased AC gene expression and venetoclax resistance. Targeting AC enhanced single-agent venetoclax cytotoxicity and the venetoclax + cytarabine combination in AML cell lines with primary or acquired venetoclax resistance. SACLAC + venetoclax was equipotent to the combination of venetoclax + cytarabine at reducing cell viability when evaluated ex vivo across a cohort of 71 primary AML patient samples. Mechanistically, SACLAC + venetoclax increased ceramide to levels that trigger a cytotoxic integrated stress response (ISR), ISR-mediated NOXA protein upregulation, mitochondrial dysregulation, and caspase-dependent cell death. Collectively, these data demonstrate the efficacy of co-targeting AC and BCL-2 in AML and rationalize targeting AC as a therapeutic approach to overcome venetoclax resistance.

cancer biology↗

bioRxiv2025

Deep-Learning on condition specific expression profiles reveal critical cytosines in gene regulation

Compared to other nucleotides, the cytosines stand as the most expressive one for gene regulation in plants due to its status as methylation-based epigenetic switch. Methylation of some of these cytosines may have higher impact on downstream genes, making them critical ones. To this date not much has been done to decipher the criticality of such cytosines. This is first such pioneering study in decoding the critical Cs where a large volume of bisulfite and RNA-seq data, including 232 WGBS and 260 corresponding RNA-seq datasets from A. thaliana and rice, have been utilized. Using a deep learning system, strong relationship was established between methylation states of cytosines in contextual manner with respect to the downstream genes expression levels. Using the same system, all the 2kb upstream regions for Arabidopsis and rice genes have been annotated for critical cytosines. Experimental validation demonstrated specific methylation changes at critical cytosines under heat stress, affecting gene expression. The universal method developed may be applied to annotate other plant genomes for critical cytosine identification. GC% similarity, rather than homology, explains gene regulatory behavior under methylation control. The resource, CritiCal-C portal, is publicly accessible, paving the way for revolutionary strategies in gene regulation and expression with minimal interventions.

bioinformatics↗

bioRxiv2025

In-dwelling microfluidic device for precise and reliable intranasal drug delivery during freely-moving behavior

Many substances/drugs are administered intranasally (IN). These include opioid overdose reversal drugs, anti-epileptic medications, migraine medications, hormone treatments, and medicines to treat/prevent allergies, colds, and flues including nasally-administered vaccines, corticosteroids, antihistamines, and decongestants. Additionally, IN administration is the preferred route of entry by users of illicit drugs. Despite the widespread use of the IN route of administration, there is no established paradigm to access this route of administration preclinically to yield precise and reliable control over delivery. This poses major gaps in therapeutic discovery/testing, establishing pharmacokinetic/pharmacodynamic relationships of therapeutics, and understanding the mechanisms of actions of therapeutics. We developed an in-dwelling microfluidic device, that, when implanted upon the nasal bone, accesses the nasal cavity to allow reliable and precise IN fluid delivery during freely-moving behavior. We validated this device, called the Nasal Access Port (NAP), to confirm it allows rapid and precise control of fluids. We further exemplified the application of the NAP for studying outcomes of IN cocaine in mice, including its pharmacokinetic profile, and both the rapid release of dopamine (DA) and behavioral effects upon IN cocaine. By achieving precise and reliable access to the IN route of administration, the NAP represents a significant methodological advance with broad applicability in the biomedical and life sciences, especially in the neuroscience, pharmacology, medicinal chemistry, and physiology domains.

pharmacology and toxicology↗

bioRxiv2025

Blue light sensor-guided opto-modulation for enhanced production of valuable metabolites in microalgae

Light sensing proteins, photoreceptor, coordinate with photosynthetic machinery, influencing both photosynthetic efficiency and several metabolic outcomes. This work deals with bottleneck issue of astaxanthin production from Chlamydomonas reinhardtii. We report a non-genetic, illumination-based strategy for biomanufacturing of astaxanthin under optimised blue light illumination in C. reinhardtii. Notably, first time ever, we identified and report astaxanthin synthesis-associated BGC in C. reinhardtii using plantiSMASH analysis. We discover experimental evidences for phototropin-modulated biosynthetic gene clusters (BGC)-mediated metabolite production in C. reinhardtii. We established that the prolonged exposure to fine-tuned blue light illumination substantially enhances the biomanufacturing of astaxanthin (1.6 times compared to red light) and pigments via the phototropin. It suggests that fine-tuned illumination conditions modulate molecular components in specific metabolite production. We are providing biochemical, genetic, transcriptomic, quantitative proteomics and systems biology evidences for opto-biomanufacturing of bioactive from green lineage via modulation of phototropin network with artificial illumination (without any genetic modification). By integrating data-driven analytics and systems biology-based computational pipeline, we elucidate the functional crosstalk between biosynthetic gene clusters with the phototropin and carotenoid metabolism synthesis pathway in the green lineage (terrestrial alga and higher plants). Thus, highlighting a new opto-biotechnology approach in metabolite production from various phototrophic organisms. These results establish a new avenue for opto-biomanufacturing strategies to generate bioactive molecules from microorganisms and other green lineages simply by illumination. Highlights O_LIOpto-biomanufacturing of astaxanthin in C. reinhardtii is achieved simply by illumination. C_LIO_LIBiosynthetic gene clusters of metabolite production are also controlled by light. C_LIO_LIPhotoreceptor-based opto-biomanufacturing of valuable bioactives is established. C_LIO_LIOpto-biomanufacturing opens avenues for bioactive production in various organisms. C_LI

biochemistry↗

bioRxiv2025

Scaled multidimensional assays of variant effect identify sequence-function relationships in hypertrophic cardiomyopathy

BackgroundAn estimated 1 in 500 people live with hypertrophic cardiomyopathy (HCM), a disease for which genetic diagnosis can identify family members at risk, and increasingly guide therapy. Mutations in the myosin binding protein C3 (MYBPC3) gene account for a significant proportion of HCM cases. However, many of these variants are classified as variants of uncertain significance (VUS), complicating clinical decision-making. Scalable methods for variant interpretation in disease-specific cell types are crucial for understanding variant impact and uncovering disease mechanisms. MethodsWe developed a scaled multidimensional mapping strategy to evaluate the functional impact of variants across a critical domain of MYBPC3. We incorporate saturation base editing at the native MYBPC3 locus, a long-read RNA sequencing-enabled assay of variant splice effects, and measurements of HCM-relevant phenotypes, including MYBPC3 abundance, hypertrophic signaling, and ubiquitin-proteasome function in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). ResultsOur multidimensional mapping strategy enabled high-resolution functional analysis of MYBPC3 variants in iPSC-CMs. Targeted transient base editing generated a comprehensive variant library at the native locus, capturing diverse variant effects on cellular HCM-relevant phenotypes. Our massively parallel splicing assay identified novel splice-disrupting variants. Integration of functional assays revealed that decreased MYBPC3 abundance is a key driver of HCM-related phenotypes. In parallel, downregulation of protein degradation was observed as a compensatory response to MYBPC3 loss of function, and novel disease mechanisms were identified for missense variants near a critical binding domain, underscoring their contribution to pathogenesis. Bayesian estimates of variant effects enable the reclassification of clinical variants. ConclusionsThis work provides a platform for extending genome engineering in iPSCs to multiplexed assays of variant effects across diverse disease-relevant cellular phenotypes, enhancing the understanding of variant pathogenicity and uncovering novel biological mechanisms that could inform therapeutic strategies.

genetics↗

bioRxiv2025

A novel molecular sexing method for Greater One-horned Rhinoceros (Rhinoceros unicornis) for reliable demographic assessments

Accurate assessment of demographic parameters is crucial for conserving endangered species. The Greater One-horned Rhinoceros (Rhinoceros unicornis), a sexually monomorphic species, lacks a validated molecular sexing protocol for diverse biological samples. This study established a multi-marker molecular sexing protocol using blood, tissue, horn, and dung samples from all wild populations in India. The optimized multiplex strategy that involves three sex-linked markers: ZF, AMESC, and TSRY, enabled simultaneous amplification of X-and Y-chromosome-specific fragments in degraded samples. Testing with 286 individual rhino samples from seven parks (5 blood, 180 tissue, seven horn, and 94 dung) led to a [~]92% (195 M: 69 F) success rate. Overall, this approach validates a molecular sexing protocol with increased reliability and fewer amplification failures, facilitating large-scale demographic studies. Future research should utilize this method to examine the sex ratio of all existing Indian rhino populations across their range, ensuring their future persistence and management.

ecology↗

bioRxiv2025

Extracellular matrix viscoelasticity regulates mammary branching morphogenesis

Structural and mechanical cues from the extracellular matrix (ECM) regulate tissue morphogenesis. Tissue development has conventionally been studied with ex vivo systems where mechanical properties of the extracellular environment are either poorly controlled in space and time, lack tunability, or do not mimic ECM mechanics. For these reasons, it remains unknown how matrix stress relaxation rate, a time-dependent mechanical property that influences several cellular processes, regulates mammary branching morphogenesis. Here, we systematically investigated the influence of matrix stress relaxation on mammary branching morphogenesis using 3D alginate-collagen matrices and spheroids of human mammary epithelial cells. Slow stress relaxing matrices promoted significantly greater branch formation compared to fast stress relaxing matrices. Branching in slow stress relaxing matrices was accompanied by local collagen fiber alignment, while collagen fibers remained randomly oriented in fast stress relaxing matrices. In slow stress relaxing matrices, branch formation was driven by intermittent pulling contractions applied to the local ECM at the tips of elongating branches, which was accompanied by an abundance of phosphorylated focal adhesion kinase (phospho-FAK) and {beta}1 integrin at the tips of branches. On the contrary, we observed that growing spheroids in fast stress relaxing matrices applied isotropic pushing forces to the ECM. Pharmacological inhibition of both Rac1 and non-muscle myosin II prevented epithelial branch formation, regardless of matrix stress relaxation rate. Interestingly, restricting cellular expansion via increased osmotic pressure was sufficient to impede epithelial branching in slow stress relaxing matrices. This work highlights the importance of stress relaxation in regulating and directing mammary branch elongation.

bioengineering↗