Biology subjects

Tiwari, A.

Publications and source records attributed to Tiwari, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A TORC1-PHR1 signaling axis regulates phosphorus starvation and immunity signaling network in Arabidopsis

The Target of Rapamycin Complex 1 (TORC1) is a crucial eukaryotic kinase that modulates growth in response to nutrient availability. Phosphorus (P) is an essential macronutrient, and its deficiency induces extensive reprogramming of growth and defense strategies in plants. This process involves Phosphate Starvation Response 1 (PHR1), a master regulator of the Phosphate Starvation Response (PSR). In this study, we identify a novel, non-canonical role for TORC1 in regulating P starvation responses in Arabidopsis. We demonstrate that P limitation activates TORC1, leading to the stabilization of PHR1. Inhibition of TORC1 increased sensitivity to P starvation, accompanied by disruption of starvation-induced transcriptional reprogramming. Additionally, our results reveal that the TORC1-PHR1 signaling axis plays a crucial role in reprogramming the expression of genes involved in the plant immune signaling network. This regulation is critical for the symbiotic association with the endophytic fungus Piriformospora indica under P starvation. These findings underscore the significant role of the TORC1-PHR1 module in orchestrating the PSR and highlight the evolutionary adaptation of TORC1 signaling pathways in plants.

plant biology↗

bioRxiv2024

Differential expression of GABARAPs in GBM renders temozolomide sensitivity in a p53-dependent manner

Glioblastoma (GBM) is one of the most debilitating and extremely aggressive tumors, with a median survival of less than a year. GBMs have high metastatic potential and frequently acquire chemoresistance. The current multimodal treatment approaches for GBM include surgical tumor resurrection, radiotherapy, and chemotherapy but these approaches leave the patient with long-term disabilities such as depletion of cognitive abilities, leukoencephalopathy, and recurrence in 6-8 months. Glioma cells are highly dependent on autophagy to survive and proliferate. Autophagy inhibition has proven to be a beneficial strategy for restricting glioma growth. However, the autophagy pathway cannot be efficiently targeted due to the lack of specific autophagy inhibitors. Understanding the vulnerabilities in autophagy gene expression can help to design better autophagy inhibitors. This study demonstrates the differential expression of GABARAP family members in low-grade glioma and GBM. Our study highlights the differential expression of GABARAP family members in response to autophagy inhibition and induction. Moreover, the knockdown of specific GABARAP family members enhanced proliferation and reduced temozolomide (TMZ) sensitivity of glial cells by decreasing the p53 expression. The selective expression pattern of GABARAP genes in Glioblastoma can be utilized to screen for patients who might respond better to temozolomide treatment. The differential expression of GABARAP family members highlights the subtle regulation of the autophagy pathway in response to environmental cues.

cancer biology↗

bioRxiv2024

Coevolving Residues Distant from the Ligand Binding Site are Involved in GAF Domain Function

Ligand binding to GAF domains regulates the activity of associated catalytic domains in a wide variety of proteins. For instance, cGMP binding to the GAFa domain of phosphodiesterase 5 (PDE5) activates the cGMP-hydrolyzing catalytic domain in the protein. However, the residues involved and the mechanism of GAF domain function are not entirely clear. Here, combining computational and experimental analysis, we show that two highly coevolving residues distant from the ligand binding site play a critical role in GAF domain allostery. Specifically, Statistical Coupling Analysis (SCA) of GAF domain sequences revealed the highest coevolution score for residues L267 and F295. Molecular dynamics (MD) simulations of both apo and holo forms of the wild type and mutant (L267A and F295A) PDE5 GAFa domains revealed significant alterations in structural dynamics and interaction with cGMP. Incorporation of the mutations in a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor, which reports a ligand-induced conformational change, revealed a change in the conformation of the GAF domain and an increase in the EC50 of cGMP-induced conformational change. Similar results were obtained regarding cGMP-induced conformational change in the full-length PDE5 and in the fluorescence of the GAF domain fluorescent protein, miRFP670nano3. Finally, structural analysis of conformers observed in MD simulations revealed a possible mechanism underlying the impact of mutations of these two coevolving residues in the PDE5 GAFa domain. Our results provide insight into the role of distant, coevolving residues in GAF domain allostery, and may aid in understanding evolution of allostery in proteins.

molecular biology↗

bioRxiv2024

Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission

Glucose has long been considered the primary fuel source for the brain. However, glucose levels fluctuate in the brain during sleep, intense circuit activity, or dietary restrictions, posing significant metabolic stress. Here, we demonstrate that the mammalian brain utilizes pyruvate as a fuel source, and pyruvate can support neuronal viability in the absence of glucose. Nerve terminals are sites of metabolic vulnerability within a neuron and we show that mitochondrial pyruvate uptake is a critical step in oxidative ATP production in hippocampal terminals. We find that the mitochondrial pyruvate carrier is post-translationally modified by lysine acetylation which in turn modulates mitochondrial pyruvate uptake. Importantly, our data reveal that the mitochondrial pyruvate carrier regulates distinct steps in synaptic transmission, namely, the spatiotemporal pattern of synaptic vesicle release and the efficiency of vesicle retrieval, functions that have profound implications for synaptic plasticity. In summary, we identify pyruvate as a potent neuronal fuel and mitochondrial pyruvate uptake as a critical node for the metabolic control of synaptic transmission in hippocampal terminals. HIGHLIGHTSO_LISerum pyruvate is taken up by the brain and efficiently oxidized in the TCA cycle. C_LIO_LIThe mitochondrial pyruvate carrier (MPC) is essential for presynaptic energy metabolism. C_LIO_LIAcetylation of the MPC complex modulates mitochondrial pyruvate uptake. C_LIO_LIMPC activity regulates the release and retrieval of synaptic vesicles in nerve terminals. C_LI

neuroscience↗

bioRxiv2024

Nerve injury downregulates δ-opioid and cannabinoid CB1 receptor genes through REST in primary sensory neurons

The transcription repressor REST in the dorsal root ganglion (DRG) is upregulated by peripheral nerve injury and promotes the development of chronic pain. However, the genes targeted by REST in neuropathic pain development remain unclear. The expression levels of 4 opioid receptor (Oprm1, Oprd1, Oprl1, Oprk1) and the cannabinoid CB1 receptor (Cnr1) genes in the DRG regulate nociception. In this study, we determined the role of REST in the control of their expression in the DRG induced by spared nerve injury (SNI) in both male and female mice. Transcriptomic analyses of male mouse DRGs followed by quantitative reverse transcription polymerase chain reaction analyses of both male and female mouse DRGs showed that SNI upregulated expression of Rest and downregulated mRNA levels of all 4 opioid receptor and Cnr1 genes, but Oprm1 was upregulated in female mice. Analysis of publicly available bioinformatic data suggested that REST binds to the promoter regions of Oprm1 and Cnr1. Chromatin immunoprecipitation analyses indicated differing levels of REST at these promoters in male and female mice. Full-length Rest conditional knockout in primary sensory neurons reduced SNI-induced pain hypersensitivity and rescued the SNI-induced reduction in the expression of Oprd1 and Cnr1 in the DRG in both male and female mice. Our results suggest that nerve injury represses the transcription of Oprd1 and Cnr1 via REST in primary sensory neurons and that REST is a potential therapeutic target for neuropathic pain.

neuroscience↗

bioRxiv2023

Investigating bacteria-phage interaction dynamics using droplet-based technology

An alarming rise in antimicrobial resistance worldwide has spurred efforts into the search for alternatives to antibiotic treatments. The use of bacteriophages, bacterial viruses harmless to humans, represents a promising approach with potential to treat bacterial infections (phage therapy). Recent advances in microscopy-based single-cell techniques have allowed researchers to develop new quantitative approaches for assessing the interactions between bacteria and phages, especially the ability of phages to eradicate bacterial pathogen populations. Here we combine droplet microfluidics with fluorescence time-lapse microscopy to characterize the growth and lysis dynamics of the bacterium Escherichia coli confined in droplets when challenged with phage. We investigated phages that promote lysis of infected E. coli cells, specifically, a phage species with DNA genome, T7 (Escherichia virus T7) and two phage species with RNA genomes, MS2 (Emesvirus zinderi) and Q{beta} (Qubevirus durum). Our microfluidic trapping device generated and immobilized picoliter-sized droplets, enabling stable imaging of bacterial growth and lysis in a temperature-controlled setup. Temporal information on bacterial population size was recorded for up to 25 hours, allowing us to determine growth rates of bacterial populations helping us uncover the extent and speed of phage infection. In the long-term, the development of novel microfluidic and single-cell techniques will expedite research towards understanding the genetic and molecular basis of rapid phage-induced lysis, preempting bacterial resistance to phages and ultimately identifying key factors influencing the success of phage therapy.

microbiology↗

bioRxiv2023

Label-free analysis of bacterial growth and lysis at the single-cell level using droplet microfluidics and object detection-oriented deep learning

Bacteria identification and counting at the small population scale is important to many applications in the food safety industry, the diagnostics of infectious diseases and the study and discovery of novel antimicrobial compounds. There is still a lack of easy to implement, fast and accurate methods to count populations of motile cells at the single-cell level. Here, we report a label-free method to count and localize bacterial cells freely swimming in microfluidic anchored picolitre droplets. We used the object detection oriented YOLOv4 deep learning framework for cell detection from bright-field images obtained with an automated Z-stack setup. The neural network was trained to recognize Escherichia coli cell morphology with an average precision of approximately 84%. This allowed us to accurately identify individual cell division events, enabling the study of stochastic bacterial growth starting from initial populations as low as one cell. This work also demonstrates the ability to study single cell lysis in the presence of T7 lytic bacterial viruses (phages). The high precision in cell numbers facilitated the visualization of bacteria-phage interactions over timescale of hours, paving the way towards deciphering phage life cycles in confined environments.

microbiology↗

bioRxiv2023

Validation of hypermethylated DNA regions found in colorectal cancers as potential aging-independent biomarkers of precancerous colorectal lesions

BackgroundWe previously identified 16,772 colorectal cancer-associated hypermethylated DNA regions that were also detectable in precancerous colorectal lesions (preCRCs) and unrelated to normal mucosal aging. We have now conducted a study to validate 990 of these differentially methylated DNA regions (DMR) in a new series of preCRCs. MethodsWe used targeted bisulfite sequencing to validate these 990 potential biomarkers in 59 preCRC tissue samples (41 conventional adenomas, 18 sessile serrated lesions), each with a patient-matched normal mucosal sample. Based on differential DNA methylation tests, a panel of (candidate) DMRs was chosen on a subset of the (our) cohort and validated on the remaining part of our cohort and (two) further publicly available datasets with respect to their stratifying potential between preCRCs and normal mucosa. ResultsStrong statistical significance for the difference in methylation levels was observed across the full set of 990 investigated DMRs. From these, a selected candidate panel of 30 DMRs correctly identified 58/59 tumors (area under the receiver operating curve: 0.998). ConclusionsThese validated DNA hypermethylation markers can be exploited to develop more accurate noninvasive colorectal tumor screening assays.

genomics↗

bioRxiv2023

Sex-dependent improvement in traumatic brain injury outcomes after liposomal delivery of dexamethasone in mice.

Traumatic Brain Injury (TBI) can have long-lasting physical, emotional, and cognitive consequences due to the neurodegeneration caused by its robust inflammatory response. Despite advances in rehabilitation care, effective neuroprotective treatments for TBI patients are lacking. Furthermore, current drug delivery methods for TBI treatment are inefficient in targeting inflamed brain areas. To address this issue, we have developed a liposomal nanocarrier (Lipo) encapsulating dexamethasone (Dex), an agonist for the glucocorticoid receptor utilized to alleviate inflammation and swelling in various conditions. In vitro studies show that Lipo-Dex were well tolerated in human and murine neural cells. Lipo-Dex showed significant suppression of inflammatory cytokines, IL-6 and TNF-, release after induction of neural inflammation with lipopolysaccharide. Further, the Lipo-Dex were administered to young adult male and female C57BL/6 mice immediately after a controlled cortical impact injury. Our findings demonstrate that Lipo-Dex can selectively target the injured brain, thereby reducing lesion volume, cell death, astrogliosis, the release of proinflammatory cytokines, and microglial activation compared to Lipo-treated mice in a sex-dependent manner, showing a major impact only in male mice. This highlights the importance of considering sex as a crucial variable in developing and evaluating new nano-therapies for brain injury. These results suggest that Lipo-Dex administration may effectively treat acute TBI.

neuroscience↗

bioRxiv2023

Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation

Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, glucose levels in the brain plummet, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1 transcriptional program that induces the expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) both in vitro and in vivo. We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by powering the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 ensures the metabolic plasticity of synaptic transmission. HighlightsO_LIGlucose deprivation drives transcriptional reprogramming of neuronal metabolism via CREB and PGC1. C_LIO_LIGlucose or food deprivation trigger the neuronal expression of mitochondrial deacetylase sirtuin 3 (Sirt3) both in vitro and in vivo. C_LIO_LISirt3 stimulates oxidative ATP synthesis in nerve terminals. C_LIO_LISirt3 sustains the synaptic vesicle cycle in the absence of glucose. C_LI

neuroscience↗

bioRxiv2023

Flat clathrin lattices are linked to metastatic potential in colorectal cancer

Clathrin assembles at the cells plasma membrane in a multitude of clathrin-coated structures (CCSs). Among these are flat clathrin lattices (FCLs), alternative clathrin structures that have been found in specific cell types, including cancer cells. Here we show that these structures are also present in different colorectal cancer (CRC) cell lines, and that they are extremely stable with lifetimes longer than 8 hours. By combining cell models representative of CRC metastasis with advanced fluorescence imaging and analysis, we discovered that the metastatic potential of CRC is associated with an aberrant membranous clathrin distribution, resulting in a higher prevalence of FCLs in cells with a higher metastatic potential. These findings suggest that clathrin organization might play an important yet unexplored role in cancer metastasis.

cancer biology↗

bioRxiv2022

Extraction and characterization of cellulose from invasive weeds from central Nepal: A potential prospect of environmental management.

The cellulose is made up of long chains of polysaccharide of glucose molecules. Microfibrils are formed when numerous hydrogen-bonded cellulose chains unite, they are extremely stiff and contribute to physical stability, due to this general ability of forming these microfibrils to form long chains, cellulose is an ideal molecule for the manufacturing of packaging materials and bioplastics. On the other hand, Invasive plant species are one of the major constituents for environmental degradation and its application seems outmost. The main purpose of this study is to extract and identify the composition of cellulose fiber and characterize the fiber of invasive plant species that could be used as a replacement for plastics and textiles in some cases. In this study, Cellulose was isolated from 6 invasive species collected in Nepals Ramechhap district using various techniques, the composition of the fiber was identified using AOAC method 973.18, ASTM method D1106-96 and ASTM method E1755-01 and characterized using FTIR spectroscopy with weight analyses. Acid hydrolysis, chlorination, alkaline extraction, and bleaching were among the chemical methods adopted. In all of the samples, there were two primary absorbance peaks. The first occurred at low wavelengths in the 700-1,800 cm-1 range, while the second occurred at higher wavelengths in the 2,700-3,500 cm-1 range.The percentage of lignin within the final sample was determined in the range of 4.4-3.1% and the percentage yield of cellulose was determined within the range of 78-62%.The study shows that the cellulose can be extracted from the taken invasive plant species and can be used for further applications.

biochemistry↗

bioRxiv2022

Enhancement of mitochondrial function fosters B cell immune memory

Differentiation of T and B cells to effector and memory cell fates are associated with extensive metabolic changes which are accompanied by altered mitochondrial dynamics. However, whether alterations in mitochondrial structure and function plays an active role in regulating effector versus memory cell fate decisions during immune responses remains unclear. Our studies here characterize changes in mitochondrial dynamics in activated B cells and show that increased mitochondrial mass and activity is a distinct feature of memory B cell lineage commitment in vivo. Using a directed screen of mitochondrial modulators, we identify mitochondrial fission inhibitor, Mdivi-1 as an agent that could enhance mitochondrial mass and function leading to augmented memory B cell differentiation. The enhanced memory B cell responses mediated by Mdivi-1, translated to more robust recall responses upon secondary antigen exposures. Moreover, Mdivi-1 when used in combination with subunit (SARS-CoV2) and inactivated (H1N1 influenza) vaccines led to remarkably improved vaccine efficacies and protection from lethal viral (H1N1) challenge. Single-cell transcriptomics revealed enhanced commitment to memory lineage differentiation in B cells following Mdivi-1 treatment. We propose that mitochondrial modulators such as Mdivi-1 are a novel class of "immune enhancers" that specifically reinforces immunological memory and could be broadly applied to improve the fidelity of immune responses and vaccine efficacies.

immunology↗

bioRxiv2022

Combinatorial encoding of odors in the mosquito antennal lobe

Among the cues that a mosquito uses to find a host for blood-feeding, the smell of the host plays an important role. Previous studies have shown that host odors contain hundreds of chemical odorants, which are detected by different receptors on the peripheral sensory organs of mosquitoes. But how individual odorants are encoded by downstream neurons in the mosquito brain is not known. We developed an in vivo preparation for patch-clamp electrophysiology to record from projection neurons and local neurons in the antennal lobe of Aedes aegypti. Combining intracellular recordings with dye-fills, morphological reconstructions, and immunohistochemistry, we identify different sub-classes of antennal lobe neurons and their putative interactions. Our recordings show that an odorant can activate multiple neurons innervating different glomeruli, and that the stimulus identity and its behavioral preference are represented in the population activity of the projection neurons. Our results provide the first detailed description of olfactory neurons in the central nervous system of mosquitoes and lay a foundation for understanding the neural basis of their olfactory behaviors.

neuroscience↗

bioRxiv2022

Structural characterization of a breast cancer-associated mutation in caveolin-1

Caveolin-1 (CAV1) is a membrane sculpting protein that oligomerizes to generate flask-shaped invaginations of the plasma membrane known as caveolae. Mutations in CAV1 have been linked to multiple diseases in humans. Such mutations often interfere with oligomerization and the intracellular trafficking processes required for successful caveolae assembly, but the molecular mechanisms underlying these defects have not been structurally explained. Here, we investigate how a breast cancer-associated mutation in one of the most highly conserved residues in CAV1, P132L, affects CAV1 structure and oligomerization. We show that P132 is positioned at a major site of protomer-protomer interactions within the CAV1 complex, providing a structural explanation for why the mutant protein fails to homo-oligomerize correctly. Using a combination of computational, structural, biochemical, and cell biological approaches, we find that despite its homo-oligomerization defects P132L is capable of forming mixed hetero-oligomeric complexes with wild type CAV1 and that these complexes can be incorporated into caveolae. These findings provide insights into the fundamental mechanisms that control the formation of homo- and hetero-oligomers of caveolins that are essential for caveolae biogenesis, as well as how these processes are disrupted in human disease.

biophysics↗

bioRxiv2021

Biomechanical and biochemical assessment of YB-1 expression in melanoma cells

Malignant melanoma is the most lethal form of skin cancer; its incidence has increased over the last five decades. Y-box binding protein 1 (YB-1) plays a prominent role in mediating metastatic behavior by promoting epithelial-to-mesenchymal transition (EMT) processes. Migratory melanoma cells exhibit two major phenotypes: elongated mesenchymal or rounded amoeboid. The actomyosin cytoskeleton is key in both phenotypes, but intermediate filaments also undergo a significant rearrangement process, switching from cytokeratin-rich to vimentin and nestin-rich network. In this study, we aimed to investigate to what extent YB-1 impacts the biomechanical (cell stiffness) and biochemical aspects of melanoma cells and their cytoskeleton. To this end, we subjected A375 YB-1 knock-out and parental cells to atomic force microscopy investigations (stiffness determination), immunolabelling, and proteome analysis. We found that YB-1 expressing cells were significantly stiffer compared to the corresponding YB-1 knock-out cell line. Proteomic analysis revealed that expression of YB-1 results in a strong co-expression of nestin, vimentin, fascin-1, and septin-9. In the YB-1 knock-out nestin was completely depleted, but zyxin was strongly upregulated. Collectively, our results showed that YB-1 knock-out acquires some characteristics of mesenchymal phenotype but lacks important markers of malignancy and invasiveness such as nestin or vimentin. We posit that there is an association of YB-1 expression with an amoeboid phenotype, which would explain the increased migratory capacity.

cancer biology↗

bioRxiv2021

Recording true oxygen reduction capacity during photosynthetic electron transfer in Arabidopsis thylakoids and intact leaves

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site- specific O2 reduction capacity of various PETC components in isolated thylakoids, the stoichiometry of oxygen production and consumption reactions, associated with H2O2 accumulation, was quantified using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to PSI is essential for H2O2 accumulation during light-induced photosynthetic electron transport process. Further leaf disc measurements provided clues that H2O2 from PETC has a potential of increasing mitochondrial respiration and CO2 release. One sentence summaryPhotosynthetically derived H2O2 only accumulates at Photosystem I and may trigger cooperation with mitochondria during stress

plant biology↗

bioRxiv2021

The landscape of extrachromosomal circular DNA in medulloblastoma

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive tumor growth, promoting high oncogene copy number, intratumoral heterogeneity, accelerated evolution of drug resistance, enhancer rewiring, and poor outcome. ecDNA has been reported in medulloblastoma (MB), the most common malignant pediatric brain tumor, but the ecDNA landscape and its association with specific MB subgroups, its impact on enhancer rewiring, and its potential clinical implications, are not known. We assembled a retrospective cohort of 468 MB patient samples with available whole genome sequencing (WGS) data covering the four major MB subgroups WNT, SHH, Group 3 and Group 4. Using computational methods for the detection and reconstruction of ecDNA1, we find ecDNA in 82 patients (18%) and observe that ecDNA+ MB patients are more than twice as likely to relapse and three times as likely to die of disease. In addition, we find that individual medulloblastoma tumors often harbor multiple ecDNAs, each containing different amplified oncogenes along with co-amplified non-coding regulatory enhancers. ecDNA was substantially more prevalent among 31 analyzed patient-derived xenograft (PDX) models and cell lines than in our patient cohort. By mapping the accessible chromatin and 3D conformation landscapes of MB tumors that harbor ecDNA, we observe frequent candidate "enhancer rewiring" events that spatially link oncogenes with co-amplified enhancers. Our study reveals the frequency and diversity of ecDNA in a subset of highly aggressive tumors and suggests enhancer rewiring as a frequent oncogenic mechanism of ecDNAs in MB. Further, these results demonstrate that ecDNA is a frequent and potent driver of poor outcome in MB patients.

cancer biology↗