Biology subjects

Rai, A. K.

Publications and source records attributed to Rai, A. K..

9 recordsLinked to original sources

bioRxiv2026

Rapid reconstitution and kinase-controlled regulation of algal pyrenoid condensates in engineered Nicotiana benthamiana

The pyrenoid is an organelle found in nearly all algae that concentrates CO2 around Rubisco to enhance photosynthetic carbon fixation. Engineering a functional pyrenoid into C plants is a promising route to improve plant photosynthetic performance and yields. However, progress has been limited by the lack of a plant platform for rapidly testing candidate components. Here, we engineered N. benthamiana to make its chloroplast Rubisco holoenzyme compatible with C. reinhardtii pyrenoid proteins. Disruption of native N. benthamiana RBCS genes and complementation with the C. reinhardtii RBCS2 generated a Rubisco-recombinant background where transient expression of the Rubisco linker protein EPYC1 was sufficient to drive the formation of Rubisco-EPYC1 condensates. Co-expression with the algal pyrenoid kinase KEY1 shifted the multi-condensate state toward a single condensate per chloroplast by altering EPYC1 phosphorylation in planta, recapitulating C. reinhardtii pyrenoid regulation. Together, these results establish engineered N. benthamiana as a tractable platform for rapidly characterizing candidate pyrenoid proteins and provide a step toward reconstructing pyrenoid architecture and regulation in plants.

plant biology↗

bioRxiv2025

Gonadal Hormone Status Dictates Neurobehavioral, Metabolic and Immunological Benefits of Ketogenic Diet in Female Mice through Gut-Metabolic-Brain Axis

The ketogenic diet (KD) is recognized for its broad therapeutic potential across metabolic, neurological, immunological, and aging-related domains; however, its efficacy and durability in females remain underexplored. This study investigated the effects of a short-term, intermittent KD on neurobehavioral function, lipid metabolism, immune response, and gut microbiota composition in sham-operated (intact) and ovariectomized (OVX, estrogen-deficient) female mice, aiming to delineate the interactions between diet and gonadal hormones. In sham females, KD reduced circulating estradiol levels and induced low-grade systemic inflammation, without producing measurable behavioral benefits. Conversely, KD exhibited reduced compulsivity, anxiety, and depression-like behaviors with improved well-being in OVX females, suggesting enhanced neurobehavioral resilience under estrogen-deficient conditions. Biochemical analyses revealed KD-induced hyperlipidemia in both groups, although the lipid load was attenuated in OVX mice. KD further decreased low-density lipoprotein cholesterol but led to uremia and histopathological evidence of hepatic steatosis and fibrosis in OVX females, despite unaltered liver function markers. 16S rRNA sequencing revealed hormone-dependent remodelling of the gut microbiota, characterised by distinct compositional shifts between the sham and OVX cohorts. Correlation analyses linked microbial alterations to improved lipid profiles and reduced anxiodepressive behaviors, implicating the gut-metabolic-brain axis in mediating the effects of KD. Collectively, these findings suggest that KD confers neurobehavioral and metabolic benefits in the absence of ovarian hormones but may also pose risks to the liver and kidneys. These results highlight ketogenic nutrition as a potential non-pharmacological strategy to mitigate menopause- and aging-associated neurobehavioral, metabolic, and immune dysfunctions through modulation of the gut microbiota.

neuroscience↗

bioRxiv2025

Kinase KEY1 controls pyrenoid condensate size throughout the cell cycle by disrupting phase separation interactions

Biomolecular condensates spatially organize cellular functions, but the regulation of their size, number, dissolution, and re-condensation is poorly understood. The pyrenoid, an algal biomolecular condensate that mediates one-third of global CO2 fixation, typically exists as one large condensate per chloroplast, but during cell division it transiently dissolves and reconfigures into multiple smaller condensates. Here, we identify a kinase, KEY1, in the model alga Chlamydomonas reinhardtii that regulates pyrenoid condensate size and number dynamics throughout the cell cycle and is necessary for normal pyrenoid function and growth. Unlike wild type, key1 mutant cells have multiple smaller condensates throughout the cell cycle that fail to dissolve during cell division. We show that KEY1 localizes to the condensates and promotes their dissolution by disrupting interactions between their core constituents, the CO2-fixing enzyme Rubisco and its linker protein EPYC1, through EPYC1 phosphorylation. We develop a biophysical model that recapitulates KEY1-mediated condensate size and number regulation and suggests a mechanism for controlling condensate position. These data provide a foundation for the mechanistic understanding of the regulation of size, number, position, and dissolution in pyrenoids and other biomolecular condensates.

cell biology↗

bioRxiv2025

Circular RNA Circ-Cdr1as modulates Macrophage phenotype and Cardiac Reparative Function by Circ-Cdr1as-miR-7-Klf4 pathway

BackgroundMechanisms of macrophage switching from pro-inflammatory to anti-inflammatory phenotypes are not well understood. Circular RNAs (circRNAs), a new class of non-coding RNAs, are implicated in immune modulation. We recently identified circ-cdr1as as a regulator of macrophage phenotype in bone marrow derived macrophages (BMDM), however, their role in immunomodulation during cardiovascular injury remains unknown. MethodsCell-specific expression levels of circ-cdr1as was determined in a mouse hearts post-myocardial infarction (MI). Circ-cdr1as was overexpressed in fluorescently labeled BMDMs and injected into the ischemic myocardium immediately following MI. Effect of AAV9-mediated systemic delivery of circ-Cdr1as on post-MI cardiac function and structure was determined. Downstream mechanisms were studied using gain and loss of function strategies. ResultsCardiac cell specific expression analysis showed significant downregulation of circ-cdr1as only in macrophages and cardiomyocytes. Overexpression of circ-cdr1as in BMDMs, injected into the ischemic myocardium retained their anti-inflammatory phenotype and significantly improved left ventricular (LV) functions and reduced infarct size. Systemic delivery of AAV9-circ-cdr1as showed similar cardiac reparative activity. Mechanistically, circ-cdr1as directly binds and sponge microRNA-7 and increases the expression of target KLF4. Loss and gain of function studies show that modulation of miR-7 and KLF recapitulates macrophage phenotypic changes. ConclusionsCirc-cdr1as plays a crucial role in regulating the anti-inflammatory phenotype of macrophages through modulation of miR-7 and its target gene KLF4. Therefore, circ-cdr1as holds potential as an anti-inflammatory regulator in tissue inflammation post-cardiac injury. Novelty and SignificanceO_ST_ABSWhat is Known?C_ST_ABSO_LIDespite continued research in elucidating mechanisms involved in cardiovascular disease and benefits of approved guideline-based therapies, the leading cause of deaths worldwide continues to be cardiovascular diseases (CVDs) with an increasing incidence of heart failure. C_LIO_LIAdvances in high-throughput RNA sequencing (RNA-seq) allowed the identification of novel transcripts such as microRNAs (miRNA), long non-coding RNAs (lncRNAs), and circular RNAs (circRNA). circular RNAs have recently emerged as promising candidates for targeted therapy due to their circular structure that confers resistance to exonucleases, their capability to regulate gene expression by modulating miRNA activity, sequester proteins by acting as protein sponges, C_LIO_LISeveral studies identified circRNAs to be differentially expressed following myocardial infarction (MI) and to play a role in immunity by contributing to the process of macrophage polarization, appropriate activation of macrophages when exposed to LPS, and inhibition of macrophage biogenesis. However, there are currently no published studies into the role of circular RNAs in the regulation of macrophage plasticity during cardiac injury. C_LI What New information Does This Article Contribute?O_LIWe provide evidence that circ-cdr1as expression is downregulated in the heart 3 days post MI and specifically in cardiomyocytes and macrophages. C_LIO_LIOur study provides promising evidence that overexpression of circ-cdr1as may be cardioprotective by reducing cardiomyocyte apoptosis, enhancing angiogenesis, limiting infarct size, increasing percentage of anti-inflammatory macrophages, and overall preserving post-MI cardiac function. C_LIO_LIMechanistically, we identified a reciprocal relationship between circ-cdr1as and miR-7 at 3 days post-MI and in naive, pro-, and anti-inflammatory macrophages indicating circ-cdr1as role as a miRNA sponge. This suggests that circ-cdr1as/miR-7/Klf4 play a crucial role in cardiac injury and macrophage phenotype. C_LI

cell biology↗

bioRxiv2023

Phosphorylation-controlled cohesion of a nuclear condensate regulates mRNA retention

Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During the cell cycle, nuclear speckles dissolve following phosphorylation of their protein components. Here, we identify the PP1 family as the phosphatases that counteract kinase-mediated dissolution. PP1 overexpression increases speckle cohesion and leads to retention of polyadenylated RNA within speckles and the nucleus. Using APEX2 proximity labeling combined with RNA-sequencing, we characterized the relationship between the cohesion of nuclear speckles and the recruitment of specific RNAs. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm, particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increased with nuclear speckle cohesion, the ratios of most mRNA species to each other were constant, indicating non-selective, or proportional, retention. We then explored whether nuclear speckle cohesion changes in response to environmental perturbations associated with changes in kinase or phosphatase activity. We found that cellular responses to heat shock, oxidative stress, and hypoxia include changes to the cohesion of nuclear speckles and mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization.

cell biology↗

bioRxiv2023

Differentiation of gram-negative intermembrane phospholipid transporter function by fatty acid saturation preference

The outer membrane of Gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. We investigated these functions using synthetic cold sensitivity (at 30 {degrees}C) caused by deletion of yhdP and fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, but not by {Delta}tamB {Delta}fadR or {Delta}ydbH {Delta}fadR. Deletion of tamB suppresses the {Delta}yhdP {Delta}fadR cold sensitivity suggesting this phenotype is related to phospholipid transport. The {Delta}yhdP {Delta}fadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not despite lower cardiolipin levels. In addition to increased cardiolipin, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the {Delta}yhdP {Delta}fadR strain. Although indirect effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential substrate transport preferences, most likely with YhdP preferentially transporting more saturated phospholipids and TamB preferentially transporting more unsaturated phospholipids. We envision cardiolipin contributing to this transport preference by sterically clogging TamB-mediated transport of saturated phospholipids. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions. Author SummaryGram-negative bacteria possess a highly impermeable outer membrane, which protects against environmental stress and antibiotics. Outer membrane phospholipid transport remained mysterious until YhdP, TamB, and YdbH were recently implicated in phospholipid transport between the inner and outer membranes of E. coli. Similar roles for YhdP and/or TamB have been suggested in both closely and distantly related gram-negative bacteria. Here, given the transporters apparent partial redundancy, we investigated functional differentiation between YhdP and TamB. Our data suggest YhdP and TamB have differential involvement with fatty acid and phospholipid metabolism. In fact, transport of higher than normal levels of cardiolipin and saturated phospholipids in the absence of YhdP and presence of TamB at a non-permissive temperature is lethal. We suggest a model where the functions of YhdP and TamB are distinguished by phospholipid transport preference with YhdP preferentially transporting more saturated phospholipids and TamB more unsaturated phospholipids. Cardiolipin headgroup specificity may contribute transport inhibition due to its bulky nature inhibiting the passage of other phospholipids. Diversification of function between YhdP and TamB provides a mechanism for regulation of phospholipid composition, and possibly the mechanical strength and permeability of the outer membrane, and so the cells intrinsic antibiotic resistance, in changing environmental conditions.

microbiology↗

bioRxiv2022

The G-protein-coupled estrogen receptor, a gene co-expressed with ERα in breast tumors, is regulated by estrogen-ERα signalling in ERα positive breast cancer cells.

PurposeThe purpose of this study was to assess the relationship between GPER, a seven transmembrane G-protein coupled estrogen receptor, and ER in breast tumors, and to make inroads into the mechanistic basis and clinical significance. MethodsTCGA-BRCA data was mined to examine the relationship between GPER and ER expression. GPER mRNA, and protein expression were analyzed in ER-positive or -negative breast tumors from two cohorts using immunohistochemistry, western blotting, or RT-qPCR. The Kaplan-Meier Plotter was employed for survival analysis. The influence of estrogen in vivo was studied by examining GPER expression levels in estrus or diestrus mouse mammary tissues, and the impact of 17{beta}-estradiol (E2) administration in juvenile or adult mice. The effect of E2, or propylpyrazoletriol (PPT, an ER agonist) stimulation on GPER expression was studied in MCF-7 and T47D cells, with or without tamoxifen or ER knockdown. ER-binding to the GPER locus was explored by analysing ChIP-seq data (ERP000380), in silico prediction of estrogen response elements, and chromatin immunoprecipitation assay. ResultsClinical data revealed significant positive association between GPER and ER expression in breast tumors. The median GPER expression in ER-positive tumors was significantly higher than ER-negative tumors. High GPER expression was significantly associated with longer overall survival of patients with ER-positive tumors. In vivo experiments showed a positive effect of E2 on GPER expression. E2 induced GPER expression in MCF-7 and T47D cells; an effect mimicked by PPT. Tamoxifen or ER-knockdown blocked the induction of GPER. Estrogen-mediated induction was associated with increased ER occupancy in the upstream region of GPER. ConclusionGPER expression is positively associated with ER in breast tumors, and a transcriptional target of the estrogen-ER signalling axis. More in-depth studies are required to establish the significance of GPER-ER co-expression, and their interplay in breast tumor development, progression, and treatment.

cancer biology↗

bioRxiv2021

Population structure and genetic diversity of chickpea germplasms

In various leguminous crops, chickpea is the fourth most important legume contributing 3.1% to the total legume production. Grains of chickpea are rich source of proteins, minerals and vitamins which makes them suitable for both food and feed. For any crop to be improved, the knowledge of genetic diversity of wild and elite cultivar is very important. Therefore among various available marker systems, molecular markers are more reliable and accurate, therefore are very commonly used for genetic diversity analysis, phylogenetic studies and cultivar identification. Due to several advantages of Inter Simple Sequence Repeat (ISSR) markers in present study we analyzed the genetic diversity, structure, cross-species transferability and allelic richness in 50 chickpea collection using 23 ISSR markers. The observed parameters such as allele number varied from 3 to 16, and PIC varied from 0.15 to 0.4988 respectively. Further, range of allele size varied from 150 to 1600 bp which shows the significance of ISSR markers for chickpea germplasms characterization. On the basis of ISSR marker genotypic data dendrogram were constructed which divides these 50 chickpea in group I and II showing the reliability of ISSR markers. Among 50 chickpea, the accession P 74-1 is in group I and rest are in group II. Further we made mini-core collection of 15 diverse chickpea and subgrouped them. Dendrogram, PCA, Dissimilarity matrix and Bayesian model based genetic clustering of 50 chickpea germplasms revealed that P 74-1,P 1883, P 1260 very diverse chickpea accession. Characterization of these diverse chickpea would help in maintenance breeding, conservation and in future could be used to develop climate resilient elite cultivar of chickpea. Utilization of these novel ISSRs markers in diversity analysis and population structure characterization of 50 chickpea germplasm suggests their wider efficacy in superior scale for molecular breeding studies in chickpea.

ecology↗

bioRxiv2020

DYRK3-Controlled Phase Separation Organizes the Early Secretory Pathway

The dual-specificity kinase DYRK3 controls formation and dissolution of several intracellular condensates thereby regulating various cell physiological processes. Here we report that DYRK3 establishes a dynamic equilibrium between condensation and dissolution of proteins associated with membranous structures of the early secretory pathway to organize membrane traffic between the ER and the Golgi complex in mammalian cells. This depends on the peripheral membrane protein Sec16A, whose N-terminal disordered region forms DYRK3-controlled liquid-like condensates on the surface of the ER and co-phase separates with multiple ER exit site components and a subset of matrix proteins specifically associated with ERGIC and cis-Golgi. Our findings support a mechanism whereby multiple interacting and differentially regulated intracellular condensates create favorable environments for directional membrane traffic in eukaryotic cells.

cell biology↗