Biology subjects

Pandey, A.

Publications and source records attributed to Pandey, A..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Loss of Mitochondrial Enoyl CoA Reductase causes elevated ceramide levels and impairs iron metabolism

In most eukaryotic cells fatty acid synthesis occurs in the cytoplasm as well as in mitochondria. However, the relative contribution of mitochondrial fatty acid synthesis (mtFAS) to the cellular lipidome of metazoans is ill-defined. Hence, we studied the function of the fly Mitochondria enoyl CoA reductase (Mecr), the enzyme required for the last step of mtFAS. Loss of mecr causes lethality while neuronal loss leads to progressive neurological defects. We observe an elevated level of ceramides, a defect in Fe-S cluster biogenesis and increased iron levels in mecr mutants. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes indicating that increased ceramides and iron metabolism are interrelated and play an important role in the pathogenesis. Mutations in human MECR cause pediatric-onset neurodegeneration and patient-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study shows an as-yet-unidentified role of mecr/MECR in ceramide and iron metabolism providing a mechanistic link between mtFAS and neurodegeneration.

neuroscience↗

bioRxiv2023

Gut barrier defects, increased intestinal innate immune response, and enhanced lipid catabolism drive lethality in N-glycanase 1 deficient Drosophila

Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of infectious versus non-infectious mechanisms on animal health in the context of barrier dysfunction is not well understood. Here, we establish that loss of Drosophila N-glycanase 1 (Pngl) leads to gut barrier defects, which cause starvation and increased JNK activity. These defects result in Foxo overactivation, which induces a hyperactive innate immune response and lipid catabolism, thereby contributing to lethality associated with loss of Pngl. Notably, germ-free rearing of Pngl mutants did not rescue lethality. In contrast, raising Pngl mutants on isocaloric, fat-rich diets improved animal survival in a dosage-dependent manner. Our data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the immune and metabolic consequences of loss of Pngl are primarily mediated through non-infectious mechanisms.

developmental biology↗

bioRxiv2023

Designer grass pea for transgene-free minimal neurotoxin-containing seeds with CRISPR-Cas9

Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid ({beta}-ODAP) has restricted its cultivation. Although various cultivars with low {beta}-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal {beta}-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the {beta}-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the backcrossed mutant lines and they displayed no phenotypic abnormalities. Interestingly, the seed {beta}-ODAP content ranged between 0.001 % - 0.002 % of dry weight which is 99 % lower than the wild-type. Together, our study reports the development of transgene-free, genome-edited grass peas with insignificant levels of {beta}-ODAP in seeds for safer food in the future.

plant biology↗

bioRxiv2023

Genetic influences vary by age and sex: Trajectories of the association of cholinergic system variants and theta band event related oscillations

To characterize systemic changes in genetic effects on brain development, the age variation of the associations of cholinergic genetic variants and theta band event-related oscillations (EROs) was studied in a sample of 2140 adolescents and young adults, ages 12 to 25 from the COGA prospective study. The theta band EROs were elicited in visual and auditory oddball (target detection) tasks and measured by EEG recording. Associations were found to vary with age, sex, task modality (auditory or visual), and scalp locality. Seven of the twenty-one muscarinic and nicotinic cholinergic SNPs studied in the analysis, from CHRM2, CHRNA3, CHRNA5, and CHRNB4, had significant effects on theta band EROs with considerable age spans for some sex-modality combination. No SNP-age-modality combination had significant effects in the same direction for males and females. Results suggest that nicotinic receptor associations are stronger before age 18, while muscarinic receptor associations are stronger after age 18.

neuroscience↗

bioRxiv2023

Genomic perspectives of SARS CoV-2 in liver disease patients with its clinical correlation: A single centre retrospective study

BackgroundSevere Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2), is a causative agent of current global pandemic of Coronavirus disease-19 (COVID-19). Due to propagated outbreak and global vaccination drive an immense immunological selection pressure has been exerted on SARS CoV-2 leading to evolution of new variants. This study was performed to compare the mutational and clinical profile of liver disease patients infected with different variants of SARS CoV-2. MethodologyThis was a single-centre, retrospective, cohort study in which clinicogenomic analysis of liver disease (LD) patients infected with SARS CoV-2 was performed. Complete demographic and clinical details were retrieved from Hospital Information System (HIS). QC-threshold passed FASTA files containing sequences from COVID-19 patients (n=174) were compared with a reference genome of SARS-CoV-2 isolate named Wuhan-Hu-1 (NCBI Reference Sequence: NC_045512.2) for mutational analysis. ResultsOut of 232 finally analysed patients 137 (59.1%) were LD-CoV (+) and 95 (40.9%) were LD-CoV(-). LD patients with comorbidities were affected more with COVID-19 (p=0.002). On comparing the outcome in the terms of mortality, LD-CoV (+) had 2.29 times (OR 2.29, CI 95%, 1.25-4.29) higher of odds of succumbing to COVID-19 (p=0.006). Multivariate regression analysis revealed, abdominal distention (p=0.05), severe COVID-19 pneumonia (p=0.046) and the change in serum bilirubin levels (p=0.005) as well as Alkaline phosphatase (ALP) levels (p=0.003) to have an association with adverse outcome in LD patients with COVID-19. In Delta (22%) and Omicron (48%) groups, Spike gene harboured maximum mutations. On comparing the mutations between LD-CoV(+/D) and LD-CoV(+/O) a total of nine genes had more mutations in LD-CoV(+/O) whereas three genes had more mutations in LD-CoV(+/D). ConclusionWe concluded that LD patients are more susceptible to COVID-19 as compared to a healthy adult with associated adverse clinical outcomes in terms of mortality and morbidity. Therefore this special group should be given priority while devising and introducing new vaccination and vaccination policies. The infection with different variants did not result in different outcome in our group of patients.

microbiology↗

bioRxiv2023

Glucose deprivation promotes pseudo-hypoxia and de-differentiation in lung adenocarcinoma

Increased utilization of glucose is a hallmark of cancer. Several studies are investigating the efficacy of glucose restriction by glucose transporter blockade or glycolysis inhibition. However, the adaptations of cancer cells to glucose restriction are unknown. Here, we report the discovery that glucose restriction in lung adenocarcinoma (LUAD) induces cancer cell de-differentiation, leading to a more aggressive phenotype. Glucose deprivation causes a reduction in alpha-ketoglutarate (KG), leading to attenuated activity of KG-dependent histone demethylases and histone hypermethylation. We further show that this de-differentiated phenotype depends on unbalanced EZH2 activity, causing inhibition of prolyl-hydroxylase PHD3 and increased expression of hypoxia inducible factor 1 (HIF1), triggering epithelial to mesenchymal transition. Finally, we identified an HIF1-dependent transcriptional signature with prognostic significance in human LUAD. Our studies further current knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to glucose deprivation and identifying novel targets to prevent the development of resistance to therapies targeting glucose metabolism.

cancer biology↗

bioRxiv2022

Production and Characterization of Monoclonal antibodies to Xenopus Proteins

Monoclonal antibodies are powerful and versatile tools that enable the study of proteins in diverse contexts. They are often utilized to assist with identifying subcellular localization and characterizing the function of target proteins of interest. However, because there can be considerable sequence diversity between orthologous proteins in Xenopus and mammals, antibodies produced against mouse or human proteins often do not recognize Xenopus counterparts. To address this issue, we refined existing protocols to produce mouse monoclonal antibodies directed against Xenopus proteins of interest. Here we describe several approaches for the generation of useful mouse anti-Xenopus antibodies to multiple Xenopus proteins and their validation in various experimental approaches. These novel antibodies are now available to the research community through the Developmental Study Hybridoma Bank (DSHB). Summary statementThe manuscript describes the generation and characterization of novel monoclonal antibodies to Xenopus laevis proteins using refined hybridoma production methods suitable for basic science research labs.

developmental biology↗

bioRxiv2022

Characterization of Integrase and Excisionase Activity in Cell-free Protein Expression System Using a Modeling and Analysis Pipeline

We present a full-stack modeling, analysis, and parameter identification pipeline to guide the modeling and design of biological systems starting from specifications to circuit implementations and parameterizations. We demonstrate this pipeline by characterizing the integrase and excisionase activity in cell-free protein expression system. We build on existing Python tools -- BioCRNpyler, AutoReduce, and Bioscrape -- to create this pipeline. For enzyme-mediated DNA recombination in cell-free system, we create detailed chemical reaction network models from simple high-level descriptions of the biological circuits and their context using BioCRNpyler. We use Bioscrape to show that the output of the detailed model is sensitive to many parameters. However, parameter identification is infeasible for this high-dimensional model, hence, we use AutoReduce to automatically obtain reduced models that have fewer parameters. This results in a hierarchy of reduced models under different assumptions to finally arrive at a minimal ODE model for each circuit. Then, we run sensitivity analysis-guided Bayesian inference using Bioscrape for each circuit to identify the model parameters. This process allows us to quantify integrase and excisionase activity in cell extracts enabling complex-circuit designs that depend on accurate control over protein expression levels through DNA recombination. The automated pipeline presented in this paper opens up a new approach to complex circuit design, modeling, reduction, and parameterization.

synthetic biology↗

bioRxiv2022

Flavonols contrary affect the interconnected glucosinolate and camalexin biosynthesis pathway in Arabidopsis thaliana

Flavonols are structurally and functionally diverse molecules playing roles in plant biotic and abiotic stress tolerance, auxin transport inhibition, pollen development, etc. Despite their ubiquitous occurrence in land plants and multifunctionality, the effect of perturbation of flavonol biosynthesis over global gene expression and pathways other than flavonoid biosynthesis has not been studied in detail. To understand the signaling role of different flavonol metabolites, herein, we used the flavonol deficient Arabidopsis thaliana loss-of-function mutant flavonol synthase1 (fls1-3) as object of study. Comparative transcriptome and metabolic profiling were used to study the effects of genetic flavonol deficiency and exogenous supplementation with flavonol derivatives (kaempferol, quercetin and rutin) on different cellular processes in the seedling. Various flavonol biosynthesis-related regulatory and structural genes were found to be up-regulated in the fls1-3 mutant which could be reversed by exogenous flavonol feeding. Our manifold comparative studies indicated the modulation of various biological processes and metabolic pathways by flavonols. Camalexin biosynthesis was found to be negatively regulated by flavonols. Interestingly, flavonols appeared to promote the accumulation of aliphatic glucosinolate through transcription factor-mediated up-regulation of biosynthesis genes. Overall, this study provides new insights into molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets and adds new knowledge to the expanding plethora of biological activity of flavonols in plants. SignificanceComparative transcriptome and metabolomic profiling of genetic flavonol deficiency and exogenous flavonol supplementation in A. thaliana seedlings, for the first-time revealed the inverse regulation of interconnected specialized metabolite pathways by flavonol aglycones, and -glycosides. Flavonols negatively regulate camalexin biosynthesis, while promoting the accumulation of aliphatic glucosinolates. Our study adds new insights into the expanding plethora of biological activity of flavonols in plants and will help to uncover the molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets.

plant biology↗

bioRxiv2022

Biochemical analysis of anthocyanin and proanthocyanidin and their regulation in determining chickpea flower and seed coat colours

Flower and seed coat colour are important agronomic traits in chickpea (Cicer arietinum L.). Cultivated chickpeas are of two types namely, desi (dark seeded, purple flower) and kabuli (light colour seeded, white flower). There has been limited information about the molecular mechanism underlying the colour variation of flower and seed coats in desi and kabuli chickpea. We profiled the anthocyanin and proanthocyanidin (PA) contents in chickpea flowers and seed coats. Tissue-specific silencing of two genes encoding a basic helix-loop-helix (CabHLH) protein and a tonoplast-localized multidrug and toxic compound extrusion (CaMATE1) transporter in a desi genotype resulted in the reduction in expressions of anthocyanin and PA biosynthetic genes and anthocyanin and PA contents in the flower and seed coat and produced flowers and seeds with kabuli characteristics. Transcriptional regulation of a subset of anthocyanin and PA biosynthetic genes by a natural CabHLH variant and transport assay of a natural CaMATE1 variant explained the association of these alleles with the kabuli phenotype. We carried out a detailed molecular characterization of these genes, and provided evidences that kabuli chickpea flower and seed colour phenotype can be derived by manipulation of single genes in a desi chickpea background. HighlightIn this study, we have defined the molecular link between flower and seed color in chickpea and identified CaMATE1 and CabHLH as the regulators of both the traits.

plant biology↗

bioRxiv2022

Interplay between R2R3 MYB-type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata)

O_LIProanthocyanidins are oligomeric flavonoid pigments that promote plant disease resistance and benefit human health. However, the transcriptional regulatory network that fine-tunes proanthocyanidin biosynthesis in banana (Musa acuminata) fruit remains poorly understood. C_LIO_LIWe characterized two proanthocyanidin-specific R2R3 MYB activators (MaMYBPA1-MaMYBPA2) and four repressors (MaMYBPR1-MaMYBPR4) to elucidate the mechanisms underlying the transcriptional regulation of proanthocyanidin biosynthesis in banana. C_LIO_LIHeterologous expression of MaMYBPA1 and MaMYBPA2 partially complemented the Arabidopsis thaliana proanthocyanidin-deficient transparent testa2 mutant. MaMYBPA1 and MaMYBPA2 interacted physically with MaMYCs and bound the promoters of the genes encoding anthocyanin synthase, leucoanthocyanidin reductase, and anthocyanidin reductase in vitro and form functional MBW complexes with MaTTG1 in A. thaliana protoplasts to transactivated these promoters in vivo. C_LIO_LIOverexpression of MaMYBPAs alone or with MaMYC in banana fruits induced proanthocyanidin accumulation and the transcription of proanthocyanidin biosynthesis- related genes. MaMYBPR repressors are also shown to interact with MaMYCs, forming repressing MBW complexes, and diminished proanthocyanidin accumulation. Interestingly the overexpression of MaMYBPA induces the expression of MaMYBPR, indicating an agile regulation of proanthocyanidin biosynthesis via the formation of competitive MBW complex. Taken together, our results reveal regulatory modules of R2R3 MYB- that fine-tune proanthocyanidin biosynthesis and offer possible targets for genetic manipulation in banana. C_LI

plant biology↗

bioRxiv2022

From Specification to Implementation: Assume-Guarantee Contracts for Synthetic Biology

We provide a new perspective on using formal methods to model specifications and synthesize implementations for the design of biological circuits. In synthetic biology, design objectives are rarely described formally. We present an assume-guarantee contract framework to describe biological circuit design objectives as formal specifications. In our approach, these formal specifications are implemented by circuits modeled by ordinary differential equations, yielding a design framework that can be used to design complex synthetic biological circuits at scale. We describe our approach using the design of a biological AND gate as a motivating, running example.

synthetic biology↗

bioRxiv2022

Hebbian and homeostatic plasticity mechanisms are segregated in sub-types of layer 5 neuron in the visual cortex

Cortical layer 5 contains two major types of projection neuron known as IB (intrinsic bursting) cells that project sub-cortically and RS (regular spiking) cells that project between cortical areas. We studied the plasticity properties of RS and IB cells in the visual cortex during the critical period for ocular dominance plasticity in mice. RS neurons exhibited synaptic depression in response to both dark exposure (DE) and monocular deprivation (MD), and their homeostatic recovery from depression was dependent on TNF. In contrast, IB cells demonstrated opposite responses to DE and MD, potentiating to DE and depressing to MD. IB cells potentiation depended on CaMKII-autophosphorylation and not TNF. IB cells showed mature synaptic properties at the start of the critical period while RS cells matured during the critical period. Together with observations in somatosensory cortex, these results suggest that differences in RS and IB plasticity mechanisms are a general cortical property. Significance StatementThe neocortex contains cells that project to different locations in the brain. In this study we show that neurons projecting to different target locations exhibit different synaptic plasticity mechanisms. Cortically projecting cells show synaptic depression and homeostatic up-regulation, subcortically projecting cells show classical Hebbian potentiation. This is important because it implies that the way a cortical neuron responds to experience and encodes information depends on the neuronal subcircuits in which it is embedded. We show that ignoring this distinction leads to erroneous conclusions regarding plasticity time-course and significance. These findings constitute an important step toward understanding how learning and memory is organized within subcircuits in the cerebral cortex.

neuroscience↗

bioRxiv2022

Ocimum sanctum, OscWRKY1, regulates phenylpropanoid pathway genes and promotes resistance to pathogen infection in Arabidopsis

WRKY transcription factor (TF) family regulates various developmental and physiological functions in plants. PAL genes encode enzymes which are involved in plant defense responses, but the direct regulation of PAL genes and phenylpropanoid pathway through WRKY TFs is not well characterized. In the present study, we have characterized an OscWRKY1 gene from O. sanctum which shows induced expression after methyl jasmonate (MeJA), salicylic acid (SA), and wounding. Recombinant OscWRKY1 protein binds to the W-box cis-element TTGAC[C/T] and activates the reporter gene in yeast. Overexpression of OscWRKY1 enhances Arabidopsis resistance towards Pseudomonas syringae pv. tomato Pst DC3000. Upstream activator sequences of PAL and C4H have identified the conserved W-box cis-element (TTGACC) in both O. sanctum and Arabidopsis. OscWRKY1 was found to interact with W-box cis-element present in the PAL and C4H promoters. Silencing of OscWRKY1 using VIGS resulted in reduced expression of PAL, C4H, COMT, F5H and 4CL transcripts. OscWRKY1 silenced plants exhibit reduced PAL activity, whereas, the overexpression lines of OscWRKY1 in Arabidopsis exhibit increased PAL activity. These results revealed that OscWRKY1 positively regulates the phenylpropanoid pathway genes and enhances the resistance against bacterial pathogen in Arabidopsis.

plant biology↗

bioRxiv2021

AtERF60 negatively regulates ABR1 to modulate salt, drought, and basal resistance in Arabidopsis

APETALA2 (AP2)/ERF family transcription factors (TFs) contribute an important function against various external cues. Our study reports AtERF60, an AP2/ERF TF, which plays a key role in regulating the ABR1 gene, leading to an altered basal resistance response in Arabidopsis. AtERF60 is induced in response to drought, salt, abscisic acid (ABA), salicylic acid (SA), and bacterial pathogen PstDC3000 infection. AtERF60 interacts with DEHYDRATION RESPONSE ELEMENTS and GCC box, indicating its ability to regulate multiple responses. The overexpressing lines of AtERF60 demonstrated increased resistance to PstDC3000 infection, whereas erf60 mutant lines showed increased susceptibility. Complementation of the erf60 mutant background exhibits no significant difference towards PstDC3000 infection compared with the WT Col-0. Microarray and qRT-PCR analysis of overexpression and mutant lines indicated that AtERF60 regulates stress-inducible genes. The induction of these differentially expressed transcripts was significantly increased in erf60 mutant lines, whereas it was reversed in wild-type lines when AtERF60 was complemented. ABR1 was one of the differentially expressed transcripts, and we discovered that AtERF60 interacts with the DRE cis-elements in the ABR1 promoter. Further, qRT-PCR expression analysis after infection with PstDC3000 in AtERF60-OX, erf60 and its complementation background suggest that the mutation in AtERF60 upregulates ABR1 activity, leading to the enhanced susceptibility towards PstDC3000. Conversely, AtERF60 overexpression suppresses ABR1 activity, strengthening the basal resistance responses of Arabidopsis.

plant biology↗

bioRxiv2021

Diverse pathogens activate the host RIDD pathway to subvert BLOS1-directed immune defense

The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1-dependent decay (RIDD) of mRNAs encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1 were resistant to infection. Non-functional Blos1 struggled to assemble the BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Blos1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal-destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus MHV also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work therefore establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.

microbiology↗

bioRxiv2021

A persistent behavioral state enables sustained predation of humans by mosquitoes

Predatory animals pursue prey in a noisy sensory landscape, deciding when to continue or abandon their chase. The mosquito Aedes aegypti is a micropredator that first detects humans at a distance through sensory cues such as carbon dioxide. As a mosquito nears its target it senses more proximal cues such as body heat that guides it to a meal of blood. How long the search for blood continues after initial detection of a human is not known. Here we show that a 5-second optogenetic pulse of fictive carbon dioxide induced a persistent behavioral state in female mosquitoes that lasted for more than 10 minutes. This state is highly specific to females searching for a blood meal and was not induced in recently blood-fed females or in males, who do not feed on blood. In males that lack the gene fruitless, which controls persistent social behaviors in other insects, fictive carbon dioxide induced a long-lasting behavior response resembling the predatory state of females. Finally, we show that the persistent state triggered by detection of fictive carbon dioxide enabled females to engorge on a blood meal mimic offered up to 14 minutes after the initial 5-second stimulus. Our results demonstrate that a persistent internal state allows female mosquitoes to integrate multiple human sensory cues over long timescales, an ability that is key to their success as an apex micropredator of humans.

neuroscience↗

bioRxiv2021

Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptors active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

biophysics↗