Biology subjects

Singh, A. P.

Publications and source records attributed to Singh, A. P..

31 records · Page 2Linked to original sources

bioRxiv2023

System analysis of differentially expressed miRNAs in hexaploid wheat display tissue-specific regulatory role during Fe deficiency response

BackgroundIron (Fe) is an essential mineral element, and its deficiency in soil largely affects crop productivity. In plants, the molecular mechanisms underlying the genetic regulation of Fe deficiency responses have yet to be well understood. Specifically, microRNA (miRNA) mediated regulation of Fe deficiency response and its regulatory network is largely elusive. In the current work, we utilized a whole genome transcriptomic approach to identify the Fe deficiency-responsive miRNAs to understand the molecular mechanisms of Fe deficiency response in wheat seedlings. The study also identifies nine novel miRNAs putatively involved in Fe deficiency response. Further, the identified miRNAs showed tissue preferences relating them to differential mechanisms against Fe deficiency. ResultsIn the present study, we performed small RNA-targeted whole genome transcriptome analysis to identify the involvement of sRNAs in Fe deficiency response. The analysis identified 105 differentially expressed miRNAs corresponding to Fe deficiency response, among them, 9 miRNAs were found to be novel in this study. Interestingly, tissue-specific regulation of Fe deficiency response also participates through miRNA-mediated regulation. We identified 17 shoot specific miRNAs and 18 root-specific miRNAs with altered expression. We validated the tissue specificity of these miRNAs by stem-loop quantitative RT-PCR. Further, an attempt was made to predict their targets to speculate their participation in Fe deficiency response. This miRNA target prediction analysis suggested a few major targets of the identified miRNAs, such as multicopper oxidases, E3 ubiquitin ligases, GRAS family, and WRKY transcription factors previously known to play key roles in Fe homeostasis. Our analysis of selected miRNAs also confirmed a temporal regulation of the response. ConclusionThe first information generated here will classify the repository of wheat miRNAs (with few novel miRNAs) for their role in Fe deficiency response. Our work provides insights into miRNA-mediated regulatory pathways during Fe deficiency.

plant biology↗

bioRxiv2022

Cis-regulatory evolution of the potassium channel gene kcnj13 during pigment pattern diversification in Danio fish

Teleost fish of the genus Danio are excellent models to study the genetic and cellular bases of pigment pattern variation in vertebrates. The two sister species Danio rerio and Danio aesculapii show divergent patterns of horizontal stripes and vertical bars that are partly caused by the evolution of the potassium channel gene kcnj13. In D. rerio, kcnj13 is required in melanophores for interactions with xanthophores and iridophores, which cause location-specific pigment cell shapes and thereby influence colour pattern and contrast. Here, we show that cis-regulatory rather than protein coding changes underlie kcnj13 evolution between the two species. D. aesculapii express lower kcnj13 levels and exhibit low-contrast patterns similar to D. rerio mutants. Our results suggest that homotypic and heterotypic interactions between the pigment cells and their shapes diverged between species by quantitative changes in kcnj13 expression during pigment pattern diversification.

evolutionary biology↗

bioRxiv2022

Trimethylamine N-oxide reduces neurite density and plaque intensity in a murine model of Alzheimer disease

BackgroundAlzheimers disease (AD) is the most common aging-associated neurodegenerative disease; nevertheless, the etiology and progression of the disease is still incompletely understood. We have previously shown that the microbially-derived metabolite trimethylamine N-oxide (TMAO) is elevated in the cerebrospinal fluid (CSF) of individuals with cognitive impairment due to AD and positively correlates with increases in CSF biomarkers for tangle, plaque, and neuronal pathology. ObjectiveWe assessed the direct impact of TMAO on AD progression. MethodsTo do so, transgenic 5XFAD mice were supplemented with TMAO for 12 weeks. ResultsOral TMAO administration resulted in significantly reduced neurite density in several regions of the brain, as assessed through quantitative brain microstructure imaging with neurite orientation dispersion and density imaging (NODDI) magnetic resonance imaging (MRI). Amyloid-{beta} plaque mean intensity was reduced, while plaque count and size remained unaltered. Proteomics analysis of the cortex revealed that TMAO treatment impacted the expression of 31 proteins (1.5-fold cut-off) in 5XFAD mice, including proteins known to influence neuronal health and amyloid-{beta} precursor protein processing. TMAO treatment did not alter astrocyte and microglial response (as determined by histological analysis) nor cortical synaptic protein expression. ConclusionThese data suggest that elevated plasma TMAO impacts AD pathology via reductions in neurite density.

neuroscience↗

bioRxiv2022

Multistage and transmission-blocking tubulin targeting potent antimalarial discovered from the open access MMV Pathogen Box

Development of resistance to current antimalarial therapies remains a significant source of concern. To address this risk, new drugs with novel targets in distinct developmental stages of Plasmodium parasites are required. In our current work, we have targeted P. falciparum Tubulin (PfTubulin) proteins which represent some of the potential drug targets for malaria chemotherapy. Plasmodial Microtubules play a crucial role during parasite proliferation, growth, and transmission, which render them highly desirable targets for the development of next-generation chemotherapeutics. Towards this, we have evaluated the antimalarial activity of Tubulin targeting compounds received from the Medicines for Malaria Venture (MMV) "Pathogen Box" against the human malaria parasite, P. falciparum (including 3D7, RKL-9 (Chloroquine resistant) and R539T (Artemisinin resistant) strains). At nanomolar concentrations, filtered out compounds exhibited pronounced multistage antimalarial effects across the parasite life cycle, including intra-erythrocytic blood stages, liver stage parasites, gametocytes and ookinetes. Concomitantly, these compounds were found to impede male gamete ex-flagellation, thus showing transmission-blocking potential of these compounds. Target mining of these potent compounds, by combining in silico, biochemical and biophysical assays, implicated PfTubulin as their molecular target, which may possibly act by disrupting microtubule assembly dynamics by binding at the interface of -{beta}Tubulin-dimer. Further, promising ADME profile of the parent scaffold supported its consideration as a lead compound for further development. Thus, our work highlights the potential of targeting PfTubulin proteins in discovering and developing next-generation, multistage antimalarial agents for treating Multi-Drug Resistant (MDR) malaria parasites. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/488926v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ae9713org.highwire.dtl.DTLVardef@40950forg.highwire.dtl.DTLVardef@1fa34f2org.highwire.dtl.DTLVardef@1aa1cae_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Optogenetic control of the Bicoid morphogen reveals fast and slow modes of gap gene regulation

Developmental patterning networks are regulated by multiple inputs and feedback connections that rapidly reshape gene expression, limiting the information that can be gained solely from slow genetic perturbations. Here we show that fast optogenetic stimuli, real-time transcriptional reporters, and a simplified genetic background can be combined to reveal quantitative regulatory dynamics from a complex genetic network in vivo. We engineer light-controlled variants of the Bicoid transcription factor and study their effects on downstream gap genes in embryos. Our results recapitulate known relationships, including rapid Bicoid-dependent expression of giant and hunchback and delayed repression of Kruppel. In contrast, we find that the posterior pattern of knirps exhibits a quick but inverted response to Bicoid perturbation, suggesting a previously unreported role for Bicoid in suppressing knirps expression. Acute modulation of transcription factor concentration while simultaneously recording output gene activity represents a powerful approach for studying how gene circuit elements are coupled to cell identification and complex body pattern formation in vivo.

developmental biology↗

bioRxiv2021

Cardiac fibroblast GSK-3α mediates adverse myocardial fibrosis via IL-11 and ERK pathway

BackgroundHeart failure is the leading cause of mortality, morbidity, and healthcare expenditures worldwide. Numerous studies have implicated Glycogen Synthase Kinase-3 (GSK-3) as a promising therapeutic target for cardiovascular diseases. GSK-3 isoforms appear to play overlapping, unique, and even opposing functions in the heart. Recently our group has identified cardiac fibroblast (CF) GSK-3{beta} as a negative regulator of fibrotic remodeling in the ischemic heart. However, the role of CF-GSK-3 in myocardial fibrosis is unknown. Methods and ResultsHerein, we employed two entirely novel conditional fibroblast-specific and tamoxifen-inducible mouse models to define the role of CF-GSK-3 in fibroblast activation and myocardial fibrosis. Specifically, GSK-3 was deleted from cardiac fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin-promoter-driven Cre recombinase. At 2 months of age, WT and KO mice were subjected to cardiac injury, and heart functions were monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 8 weeks post-injury. In both settings, GSK-3 deletion restricted fibrotic remodeling and improved cardiac function. To investigate underlying mechanisms, we examined the effect of GSK-3 deletion on myofibroblast transformation and pro-fibrotic TGF{beta}1-SMAD3 signaling in vitro. A significant reduction in cell migration, collagen gel contraction, and -SMA expression in TGF{beta}1 treated GSK-3 KO MEFs confirmed that GSK-3 is required for myofibroblast transformation. Surprisingly, GSK-3 deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK-3 is SMAD3 independent. To further delineate the underlying mechanism, total proteins were isolated from CFs of WT and KO animals at 4 weeks post-injury, and kinome profiling was performed by utilizing PamStation(R)12 high throughput microarray platform. The kinome analysis identified the downregulation of RAF family kinase activity in GSK3-KO-CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Importantly, flow cytometric analysis of CFs confirmed a significant decrease in pERK levels in KO mice. Additionally, our in vitro studies demonstrated that GSK-3 deletion prevented TGF{beta}1 induced ERK activation thereby validating our findings from kinome analysis. Interestingly, IL-11, a fibroblast specific downstream effector of TGF{beta}1, was very low in GSK-3 KO MEFs as compared to WT and ERK inhibition further reduced IL-11 expression in them. All these results indicate that GSK-3 mediates pro-fibrotic response in the injured heart through IL-11 and ERK pathway. ConclusionCF-GSK-3 plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.

molecular biology↗

bioRxiv2021

SLTRiP induces long lasting and protective T-cell memory response

Major developments have been made in the past many years to characterize and explore potential vaccine candidates that can induce host immune responses against parasite. These advances were based on the fact that the induction of host immune responses could effectively target parasite at different stages of its life cycle and thus, abrogate Plasmodium infections. The role of T-cells against malaria comes from initial studies on rodents showing these cells could inhibit parasite development during pre-erythrocytic stages. Since then, the importance of the cellular immune responses against malaria has been increasingly emphasized, especially for vaccine development against pre-erythrocytic stages. Previous work in our laboratory has confirmed that SLTRiP confers protection against the pre-erythrocytic stage of Plasmodium growth in rodents. Here we report that the protection is mainly due to cell mediated immune responses and PbSLTRiP specific cellular memory responses could be efficiently recalled in mice challenged with P. berghei parasites even after a year following immunization. Our results thereby, highlight the role of the T cell response involved in protection. Characterization of T-cells by intracellular cytokine staining (ICS) revealed that the induced T cells were polyfunctional and involved in secretion of pro-inflammatory cytokines which mediate anti-parasitic activity. The findings contribute to our understanding of the immunological mechanisms underlying the protective vaccines.

immunology↗

bioRxiv2020

Repurposing Nintedanib for Pathological Cardiac Remodeling and Dysfunction

BackgroundHeart Failure (HF) is the leading cause of death worldwide. Myocardial fibrosis, one of the clinical manifestations implicated in almost every form of heart disease, contributes significantly to HF development. However, there is no approved drug specifically designed to target cardiac fibrosis. Nintedanib (NTB) is an FDA approved tyrosine kinase inhibitor for idiopathic pulmonary fibrosis (IPF) and chronic fibrosing interstitial lung diseases (ILD). The favorable clinical outcome of NTB in IPF patients is well established. Furthermore, NTB is well tolerated in IPF patients irrespective of cardiovascular comorbidities. However, there is a lack of direct evidence to support the therapeutic efficacy and safety of NTB in cardiac diseases. Methods and ResultsWe examined the effects of NTB treatment on cardiac fibrosis and dysfunction using a murine model of HF. Specifically, 10 weeks old C57BL/6J male mice were subjected to Transverse Aortic Constriction (TAC) surgery. NTB was administered once daily by oral gavage (50mg/kg) till 16 weeks post-TAC. Cardiac function was monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 16 weeks post-TAC. In the control group, systolic dysfunction started developing from 4 weeks post-surgery and progressed till 16 weeks. However, NTB treatment prevented TAC-induced cardiac functional decline. In another experiment, NTB treatment was stopped at 8 weeks, and animals were followed till 16 weeks post-TAC. Surprisingly, NTBs beneficial effect on cardiac function was maintained even after treatment interruption. NTB treatment remarkably reduced cardiac fibrosis as confirmed by Massons trichome staining and decreased expression of collagen genes (COL1A1, COL3A1). Compared to TAC group, NTB treated mice showed lower HW/TL ratio and cardiomyocyte cross-sectional area. Our in vitro studies demonstrated that NTB prevents myofibroblast transformation, TGF{beta}1-induced SMAD3 phosphorylation, and production of fibrogenic proteins (Fibronectin-1). However, NTB significantly altered vital signaling pathways in both, isolated fibroblast and cardiomyocytes, suggesting that its biological effect and underlying cardiac protection mechanisms are not limited to fibroblast and fibrosis alone. ConclusionOur findings provide a proof of concept for repurposing NTB to combat adverse myocardial fibrosis and encourage the need for further validation in large animal models and subsequent clinical development for HF patients.

pathology↗

bioRxiv2020

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

molecular biology↗

bioRxiv2020

The Argo: A 65,536 channel recording system for high density neural recording in vivo

Here we demonstrate the Argo System, a massively parallel neural recording system based on platinum-iridium microwire electrode arrays bonded to a CMOS voltage amplifier array. The Argo system is the highest channel count in vivo neural recording system built to date, supporting simultaneous recording from 65,536 channels, sampled at over 32 kHz and 12-bit resolution. This system is designed for cortical recordings, compatible with both penetrating and surface microelectrodes. We have validated this system by recording spiking activity from 791 neurons in rats and cortical surface Local Field Potential (LFP) activity from over 30,000 channels in sheep. While currently adapted for head-fixed recording, the microwire-CMOS architecture is well suited for clinical translation. Thus, this demonstration helps pave the way for a future high data rate intracortical implant.

neuroscience↗

bioRxiv2019

Supression of Perforin-like Protein pores inhibit Plasmodium multistage-growth, transmission and erythrocyte senescence

The pore forming Plasmodium perforin like proteins (PPLP), expressed in all stages of the parasite life cycle are central drivers for host interactions critical for completion of parasite life cycle and high transmission rates. The high sequence similarity in the central membrane attack complex/ perforin (MACPF) domain and consequent functional overlaps defines them as an attractive target for the development of multi-stage antimalarials. Herein we evaluated the mechanism of pan active function of central, highly conserved region of PPLPs, MACPF domain (PMD) and inhibitory potential of specifically designed anti-PMD chemo. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to haemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence at 48 hours which can account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing anemia during malaria infection. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress of merozoites and protecting against erythrocyte senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage and transmission-blocking potential of these inhibitors. Additionally, the erythrocyte senescence protective potential of PMD inhibitors can be used to occlude PPLPs mediated severe malarial anemia. Further these inhibitors can be developed with a potential to protect against severity of the disease.\n\nAuthor SummaryMalaria continues to be a major global health threat despite of several exciting improvements in the treatment and prevention of the disease. One of the major concerns in the development of therapy is the emergence of the drug resistance. But for the efficient treatment regime, targeting multiple stages including host and vector would serve as an ideal therapy. Perforin like proteins (PLPs) are eukaryotic pore forming proteins that are highly conserved in the apicomplexan parasites. These play crucial roles in entry and exit of parasites from the host cells and establish infection at multiple stages of Plasmodium spp. life cycle. Understanding the mechanism of pore formation by smaller, functional, pan-active scaffold of PLPs can serve as a target for development of cross stage protection. Here, using various biochemical, biophysical and pharmacological evidences, we validate the activity and characterize the pore formation of PLPs on erythrocytes. Further, our specifically designed inhibitors could restrict this pore formation and impede the exit/entry of the parasites. Moreover, these inhibitors could also exert multiple stage inhibition and rescue the uninfected erythrocytes from death. Together, this study highlights the mechanism of pore formation by PPLPs and evaluates their potential for the development of pan-active inhibitors to provide both symptomatic and transmission blocking cure for malaria.

molecular biology↗

bioRxiv2018

OsJAZ9 overexpression improves potassium deficiency tolerance in rice by modulating jasmonic acid levels and signaling

Potassium (K) which makes around 2-10% of plants total dry biomass, when become deficient, makes the plants highly susceptible to both abiotic and biotic stresses. Recent evidences suggest overlapping transcriptional responses to K deficiency and Jasmonate (JA) treatment in plants. However, a link between these responses was missing. Notably, K deficiency and JA application produce similar phenotypic and transcriptional responses. Here, we used molecular, physiological and morphological studies to analyze the role of OsJAZ9 in JA homeostasis, K deficiency and sheath blight resistance. We raised OsJAZ9 overexpression, knockdown, translational reporter and C-terminal deleted translational reporter lines in rice to establish the role of JA signaling in K ion homeostasis and OsJAZ9 as a critical component of JA signaling for K deficiency response. OsJAZ9 overexpression and knockdown provide K deficiency tolerance and sensitivity, respectively, by modulating various K transporters and root system architecture. Furthermore, RNA Seq and JA profiling revealed an elevation of JA responsive genes and JA levels in OsJAZ9 OE lines under K deficiency. Our data provide clear evidence on the crucial role of JAZ repressor, OsJAZ9 in improving K deficiency tolerance in rice by altering JA levels and signaling.

plant biology↗

bioRxiv2017

ALKALs are in vivo ligands for ALK family Receptor Tyrosine Kinases in the neural crest and derived cells.

Mutations in Anaplastic Lymphoma Kinase (ALK) are implicated in somatic and familial neuroblastoma, a paediatric tumour of neural crest-derived tissues. Recently, biochemical analyses have identified secreted small ALKAL proteins (FAM150, AUG) as potential ligands for human ALK and the related Leukocyte Tyrosine Kinase (LTK). In the zebrafish Danio rerio, DrLtk, which is similar to human ALK in sequence and domain structure, controls the development of iridophores, neural crest-derived pigment cells. Hence, the zebrafish system allows studying Alk/Ltk and Alkals involvement in neural crest regulation in vivo. Using zebrafish pigment pattern formation, Drosophila eye patterning, and cell culture-based assays, we show that zebrafish Alkals potently activate zebrafish Ltk and human ALK driving downstream signalling events. Overexpression of the three Dr Alkals cause ectopic iridophore development whereas loss of function alleles lead to spatially distinct patterns of iridophore loss in zebrafish larvae and adults. alkal loss of function triple mutants completely lack iridophores and are larval lethal as is the case for ltk null mutants. Our results provide the first in vivo evidence of (i) activation of ALK/LTK family receptors by ALKALs and (ii) an involvement of these ligand-receptor complexes in neural crest development.

developmental biology↗