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Thakur, V.

Publications and source records attributed to Thakur, V..

15 recordsLinked to original sources

Enhancement of gut-microbiome-based classification of prodromal Parkinson's Disease cases by hyperparameter tuning, integration of multimodal data, and meta-analysis.

Parkinson's Disease (PD) is often preceded by a prodromal phase marked by non-motor symptoms such as REM Sleep Behaviour Disorder (RBD), gastrointestinal dysfunction, and olfactory impairment. Mounting evidence implicates gut microbiota in the early pathophysiology of PD. In this study, we aimed to enhance the classification of prodromal PD using gut microbiota profiles, with a focus on model performance and biomarker identification. Publicly available gut microbiome datasets of individuals with RBD and healthy controls were analyzed. Following batch correction and genus-level filtering, we identified six taxa significantly depleted in both RBD and PD groups, all belonging to short-chain fatty acid (SCFA)-producing genera. Hyperparameter tuning on the testing set achieved an AUC of 0.889. When combining microbial and clinical features, the model achieved a peak mean AUC of 0.917. Feature selection identified 11 top-ranked features, yielding an AUC of 0.849. Using only the six differentially abundant SCFA-producing taxa, the model retained a strong predictive capacity with a mean AUC of 0.853. These results demonstrate the utility of gut microbiota, particularly SCFA-producing taxa, in the early identification of prodromal PD. Integration with select clinical features further improves model performance, supporting their potential role in early diagnostic frameworks.

bioinformatics↗

The lymphangiogenic Ccbe1-Adamts-Vegfc axis shapes cerebrovascular regionalization via selective patterning of fenestrated vascular beds

Organ development and function require regional specialization of vascular networks, yet the underlying molecular mechanisms remain poorly understood. Here, we identify a non-canonical role for the lymphangiogenic Ccbe1-Adamts-Vegfc axis in shaping cerebrovascular regionalization in zebrafish. A ccbe1 knock-in reporter revealed highly restricted expression in the developing brain, with its genetic loss causing early vascularization defects at the pineal gland-choroid plexus interface. Simultaneous Vegfa signaling deficiency markedly exacerbated these regional vascular defects, accompanied by disrupted blood flow, local hemorrhage, and choroid plexus epithelial disorganization, while largely preserving neighboring barrier-forming vessels. Loss of vegfc closely phenocopied ccbe1 mutant defects, with fenestrated vascular patterning requiring both Vegfr2 and Vegfr3 signaling, whereas disruption of adamts3 and adamts14 indicated region-specific requirements for Vegfc-activating proteases. Conversely, pineal gland-specific overexpression of activated Vegfc partially rescued the vascularization defects. Collectively, these findings suggest that spatially restricted Ccbe1-Adamts-Vegfc activity provides angiogenic signaling specificity enabling fenestrated cerebrovascular patterning and regionalization.

developmental biology↗

Genomic Footprints of Bottlenecks, Isolation, and Inbreeding: A Case Study of Two Vulture Cohorts in India

Genomic erosion as a manifestation of small effective population size (Ne) and consanguinity subverts long-term perpetuation of threatened species by compromising their adaptive potential; however, the integration of genomics remains limited in applied conservation efforts to guide priorities. This study combines non-invasive sampling, double-digest Restriction site-associated DNA sequencing (ddRAD), and population-genomic analyses to assess genetic health in two vulture assemblages-mixed wild enclosure and captive breeding cohorts. Both the geographical locations exhibit signs of populations in distress: low genetic diversity and abundant intermediate-length runs of homozygosity (RoH), consistent with long-term reduced Ne plus recent demographic isolation. Our demographic model runs favoured ancient migration (AM) topology characterised by an ephemeral window of gene flow, taken over by a prolonged population separation period. The mutation quantification results from approximately 59,000 outgroup-polarised SNPs reveal higher additive burden and more homozygous-derived sites in BKN. However, this was later traced to low-impact and non-coding variants rather than a surge in the loss-of-function (LoF) alleles. The data support a genomic profile that carries an elevated risk from polygenic/aggregate deleterious burden in BKN despite a scarcity of high-impact mutations. By highlighting the disconnect between genetic resilience and demographic recovery, our results accentuate the need to incorporate genomics-informed inbreeding and monitoring programs, while also focusing on reducing anthropogenic mortality with genetic augmentation.

genomics↗

Targeting matrix metalloproteinase-14 disrupts DNA repair and reduces viability in adrenocortical carcinoma

BackgroundAdrenocortical carcinoma (ACC) is a rare and aggressive endocrine cancer with limited treatment options and poor prognosis. Identifying novel therapeutic targets requires understanding the molecular drivers of ACC progression and establishing translational models for preclinical validation. ResultsMatrix metalloproteinase-14 (MMP-14) is the most highly expressed MMP in ACC, and high MMP-14 expression is associated with worse overall and disease-free survival. We demonstrate that MMP-14 is essential for ACC cell survival and serves an unexpected role in maintaining genome stability. Genetic silencing or pharmacologic inhibition of MMP-14 significantly reduced viability in both NCI-H295R cells and patient-derived tumor organoids (PTOs). MMP-14 knockdown induced CHK1 activation and S-phase checkpoint arrest. Mechanistically, MMP-14 translocates to the nucleus and binds to chromatin following DNA damage induced by ionizing radiation or cisplatin. Loss of MMP-14 resulted in accumulation of DNA double-strand breaks, as evidenced by increased {gamma}H2AX foci, and impaired non-homologous end joining (NHEJ)-mediated repair. ConclusionsThese findings reveal a novel nuclear function for MMP-14 in DNA repair and identify MMP-14 as a promising therapeutic target in ACC. Targeting MMP-14 may sensitize ACC tumors to DNA-damaging chemotherapy by impairing the repair of therapy-induced lesions.

cancer biology↗

The abundance of potential B12 producers in gut microbiome is inversely associated with serum vitamin B12 levels: A putative role of Host-microbiome feedback regulation

BackgroundThe human gut microbiome is a natural source of essential micronutrients like B-vitamins, which are utilized by both the host and other community members. The prevalence and abundance of known B-vitamin producers and B-vitamin biosynthesis pathways have already been reported in gut microbiome cohorts of a few countries including India. ObjectiveTo test whether the presence of B-vitamin producers/biosynthetic pathways translates into serum B-vitamin levels, taking B12 as a case example. MethodsFecal samples were collected from non-deficient (serum B12 level > 210 pg/mL, n=29) and B12 deficient (serum B12 level < 210 pg/mL, n=30) children from a tribal region of central India. Whole metagenomic DNA was extracted, sequenced, and analyzed for taxonomic profiling and diversity comparisons. Differentially abundant taxa between two groups were identified. The prevalence and abundance of potential B12 producers were compared, and their association with serum B12 level was established. ResultsA comparison of within-sample diversity between the two groups didnt show any difference; however, between-sample diversity was significantly less in the B12 deficient group. Differential abundance testing also showed different microbiome structure in the B12 deficient group, where an increased abundance of B12 transporter-carrying Bacteroides thetaiotaomicron, a few pathogenic species, and ten known B12 producers was observed. Potential B12 producers were also significantly prevalent and abundant in the deficient group. Their cumulative abundance was also significantly higher in the deficient group and showed a negative association with serum B 12 levels. ConclusionA higher abundance of potential B12 producers in the deficient group suggested an adaptive mechanism by the gut microbiome to meet the communitys B12 requirements, by selectively promoting the growth of B12 producers.

genomics↗

A dynamin-like protein in the human malaria parasite plays an essential role in mitochondrial homeostasis and fission during asexual blood stages

Malaria parasite harbors a single mitochondrion and its proper segregation during the parasite multiplication is crucial for propagation of the parasite within the host. Mitochondrial fission machinery consists of a number of proteins that associate with mitochondrial membrane during segregation. Here, we have identified a dynamin-like protein in P. falciparum, PfDyn2, and deciphered its role in mitochondrial division, segregation and homeostasis. GFP targeting approach combined with high resolution microscopy studies showed that the PfDyn2 associates with mitochondrial membrane to form a clip/hairpin loop like structure around it at specific sites during mitochondrial division. The C-terminal degradation tag mediated inducible knock-down (iKD) of PfDyn2 resulted in significant inhibition of parasite growth. PfDyn2-iKD hindered mitochondrial development and functioning, decreased mtDNA replication, and induced mitochondrial oxidative-stress, ultimately causing parasite death. Further, treatment of parasites with dynamin specific inhibitors disrupted the recruitment of PfDyn2 on the mitochondria, blocked mitochondrial development, and induced oxidative stress. Regulated overexpression of a phosphorylation mutant of PfDyn2 (Ser-612-Ala) had no effect on the recruitment of PfDyn2 on the mitochondria; normal mitochondrial division and parasite growth showed that phosphorylation/dephosphorylation of this conserved serine residue (Ser612) may not be responsible for regulating recruitment of PfDyn2 to the mitochondrion. Overall, we show essential role of PfDyn2 in mitochondrial dynamics and fission as well as in maintaining its homeostasis during asexual cycle of the parasite.

cell biology↗

Evolutionary approaches predicted changes in transcription factors and cis-elements that associate with Kranz anatomy development in maize

C4 plants exhibit higher photosynthetic efficiency under high temperatures and low water availability due to their unique anatomical and biochemical adaptations, but the evolutionary mechanisms and regulatory components underlying their Kranz development remain partly known. If C4 traits evolved through adaptive changes, its developmental regulators should show detectable evolutionary signatures in C4 lineages. We investigated the adaptive evolutionary changes in both protein sequences and cis-motifs upstream of candidate genes differentially expressed during Kranz anatomy establishment in C4 grasses. We identified signatures of convergent evolution in candidate genes and their upstream regions by comparing C4 orthologs with their C3 counterparts, and enriched non-syntenic motifs present upstream of C4 orthologs through phylogeny-aware analysis. Out of 191 candidate genes analyzed, ten orthogroups containing maize orthologs of EREB160, DOF11, bHLH116, MADS9, bHLH33/bHLH105, SCRO1/SCR2, CADTFR3, THX8, C3H28, and SHR1/SHR2 showed positive selection pressure specific to C4 orthologs. Among them, DOF11, SCRO1/SCR2, and SHR1/SHR2 have previously been implicated in Kranz regulation by previous studies. Additionally, we identified a conserved position mutation present in bHLH116 across C4 lineages, which may influence its DNA-binding domain. Moreover, we identified 39 annotated non-syntenic/C4-shifted DNA motifs, upstream of 28 gene orthologs, enriched in C4 species. Notably, genes with these motifs did not intersect with the genes under positive selection, suggesting these pathways evolved separately without much crosstalk, nevertheless, a few putative regulatory-network-motifs were observed. These motifs are potential additions to the limited Kranz-specific motifs and can aid in predicting new putative regulators after experimental validation. Significance statementC4 plants evolved as an adaptive response through anatomical and biochemical changes in their C3 relatives. Unlike the biochemical changes, the regulators/drivers of anatomical changes and the nature of underlying changes remains only partially understood. Using signatures of adaptive evolution, the current study screened candidate regulatory factors and sequences, including a few previously known and many novel candidates. These findings complement the existing experimental knowledge and advance the discovery of the gene regulatory network underlying the anatomical changes associated with C4 evolution.

plant biology↗

A Novel AI-Designed Antimicrobial Peptide Synergistically Potentiates Aminoglycosides against Colistin- and Carbapenem-Resistant Acinetobacter baumannii

The urgent necessity for new antibiotics becomes glaringly evident with the relentless rise of multidrug-resistant (MDR) Acinetobacter baumannii in clinical environments, where its infections lead to alarmingly high mortality rates. Antimicrobial peptides (AMPs) represent a promising novel option to combat nosocomial infections caused by MDR A. baumannii. In this study, six novel synthetic peptides were designed through generative artificial intelligence (AI) and synthesized for further experiments. Peptides AIG-R1, AIG-R4, and AIG-R5 showed potent broad-spectrum antibacterial activity against Gram positive and Gram negative pathogens. One of the peptides, AIG-R5, was effective even against colistin and carbapenem-resistant strains of A. baumannii, prevented biofilm formation, and eradicated established biofilms by 60%. Notably, AIG-R5 enhanced the activity of different antibiotics and was found to exhibit synergistic activity with antibiotics from the Aminoglycoside class. The combination of AIG-R5 and Tobramycin at 1/8xMIC and 1/4xMIC effectively reduced pre-formed biofilms of carbapenem resistant A. baumannii more than either component alone, as documented by confocal laser scanning microscopy (CLSM). Significant dose reduction and negligible cytotoxicity exhibited by AIG-R5 with aminoglycosides further encourages evaluation of the combinations therapeutic potential in vivo against MDR A. baumannii infections.

microbiology↗

Dual role of an essential HtrA2/Omi protease in the human malaria parasite: maintenance of mitochondrial homeostasis and induction of apoptosis like cell death under cellular stress

HtrA family of serine protease is known to play role in mitochondrial homeostasis as well as in the programmed cell death. Mitochondrial homeostasis and metabolism are crucial for survival and propagation of malaria parasite within the host. Here we have functionally characterized a Plasmodium falciparum HtrA2 (PfHtrA2) protein, which harbours trypsin like protease activity that can be inhibited by its specific inhibitor, ucf-101. Transgenic parasite line was generated, using HA-glmS C-terminal tagging approach, for localization as well as for inducible knockdown of PfHtrA2. The PfHtrA2 was localized in the parasite mitochondrion during asexual life cycle. Genetic ablation of PfHtrA2 caused significant parasite growth inhibition, decreased replication of mtDNA, increased mitochondrial ROS production, caused mitochondrial fission/fragmentation, and hindered parasite development. However, ucf-101 treatment had no effect on the parasite growth, suggesting non-protease/chaperone role of PfHtrA2 in the parasite. Under cellular stress conditions, inhibition of PfHtrA2 by ucf-101 reduced activation of the caspase like protease as well as parasite cell death, suggesting involvement of protease activity of PfHtrA2 in apoptosis like cell death in the parasite. Under these cellular stress conditions, the PfHtrA2 gets processed but remains localized in the mitochondrion, suggesting that it acts within the mitochondrion by cleaving intra-mitochondrial substrate(s). This was further supported by trans-expression of PfHtrA2 protease domain in the parasite cytosol, which was unable to induce any cell death in the parasite. Overall, we show specific role of PfHtrA2 in maintaining mitochondrial homeostasis as well as in regulating the stress induced cell death.

cell biology↗

Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

molecular biology↗

A Plasmodium falciparum lysophospholipase regulates fatty acid acquisition for membrane biogenesis to enable schizogonic asexual division

Phospholipid metabolism is crucial for membrane biogenesis and homeostasis during the intracellular life cycle of Plasmodium falciparum. To generate large amounts of phospholipids required during blood stages, the parasite massively scavenge, recycle and reassemble host lipids. P. falciparum possesses an unusual large number of lysophospholipases. However, their functional roles and importance remain to be elucidated. Here, we functionally characterized one of P. falciparum lysophospholipase (PfLPL3) (Gene ID PF3D7_1476800), to reveal its critical role in parasite propagation during asexual blood stages. We generated a transgenic parasite line using GFP-glmS C-terminal tagging approach, for localization as well as inducible knockdown of PfLPL3. PfLPL3 displayed a dynamic localization throughout asexual stages, mainly localizing in the host parasite interface: parasitophorous vacuole space and expanding into the tubulovesicular network within the host cell. Inducible knock-down of PfLPL3 hindered normal intraerythrocytic cycle, specifically causing disruption in parasite development from trophozoites to schizont, as well as reduction in number of merozoites progenies. Thus, down-regulation of PfLPL3 significantly inhibited parasite growth suggesting its critical role for proper parasite propagation during blood stages. Detailed lipidomic analyses showed that PfLPL3 generates fatty-acids for the synthesis of neutral lipids DAG and TAG, whilst controlling the timely synthesis of phospholipids that are crucial for membrane biogenesis required for merozoite development during asexual cycle. Setting up an in vitro activity based screening of Malaria Box allowed identification of specific inhibitors of PfLPL3 having potent parasitical efficacies. These compounds are pertinent both as anti-malarial drug candidates and chemical tools specifically targeting membrane biogenesis during asexual blood stages.

microbiology↗

Implicit and explicit learning of Bayesian priors differently impacts bias during perceptual decision-making

Knowledge without awareness, or implicit knowledge, influences a variety of behaviors. It is unknown however, whether implicit knowledge of statistical structure informs visual perceptual decisions or whether explicit knowledge of statistical probabilities is required. Here, we measured visual decision-making performance using a novel task in which humans reported the orientation of two differently colored translational Glass patterns; each color associated with different orientation probabilities. The task design allowed us to assess participants ability to learn and use a general orientation prior as well as a color specific feature prior. Classifying decision-makers based on a questionnaire revealed that both implicit and explicit learners implemented a general orientation bias by adjusting the starting point of evidence accumulation in the drift diffusion model framework. Explicit learners additionally adjusted the drift rate offset. When subjects implemented a stimulus specific bias, they did so by adjusting primarily the drift rate offset. We conclude that humans can learn priors implicitly for perceptual decision-making and depending on awareness implement the priors using different mechanisms.

neuroscience↗

A role for neutral variation in the evolution of C4 photosynthesis

Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C4 photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C4 photosynthesis evolved from ancestral C3 photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C4 cycle was neutral in C3 plants. This neutrality would enable populations of C3 plants to maintain genotypes with expression levels of C4 enzymes analogous to those in C4 species and thus enable rapid assembly of a functional C4 cycle from naturally occurring genotypes given shared environmental selection. Here we show that there is substantial natural variation in expression of genes encoding C4 cycle enzymes between natural accessions of the C3 plant Arabidopsis thaliana. We further show through targeted transgenic experiments in the C3 crop Oryza sativa, that high expression of the majority of C4 cycle enzymes in rice is neutral with respect to growth, development, biomass and photosynthesis. Thus, substantial variation in the abundance and activity of C4 cycle enzymes is permissible within the limits of operation of C3 photosynthesis and the emergence of component parts of this complex convergent trait can be facilitated by neutral variation.

plant biology↗

A lysophospholipase plays role in generation of neutral-lipids required for hemozoin formation in malaria parasite

Phospholipid metabolism is crucial for membrane dynamics in malaria parasites during entire cycle in the host cell. Plasmodium falciparum harbours several members of phospholipase family, which play key role in phospholipid metabolism. Here we have functionally characterized a parasite lysophospholipase (PfLPL1) with a view to understand its role in lipid homeostasis. We used a regulated fluorescence affinity tagging, which allowed endogenous localization and transient knock-down of the protein. PffLPL1localizes to dynamic vesicular structures that traffic from parasite periphery, through the cytosol to get associated as a multi-vesicular neutral lipid rich body next to the food-vacuole during blood stages. Down-regulation of the PfLPL1 disrupted parasite lipid-homeostasis leading to significant reduction of neutral lipids in lipid-bodies. This hindered conversion of heme to hemozoin, leading to food-vacuole abnormalities, which in turn disrupted parasite development cycle and significantly inhibited parasite growth. Detailed lipidomic analyses of inducible knock-down parasites confirmed role of PfLPL1 in generation of neutral lipid through recycling of phospholipids. Our study thus suggests a specific role of PfLPL1 to generate neutral lipids in the parasite, which are essential for parasite survival.\n\nImportancePresent study was undertaken with a view to understand the functional role of a unique lipase (lysophopholipase, PfLPL1) of the human malaria parasite. We utilized genetic approaches for GFP tagging as well as to knock-down the target protein in the parasite. Our studies showed that PfLPL1 associates closely with the lysosome like organelle in the parasite, the food-vacuole. During the blood stage parasite cycle, the food-vacuole is involved in degradation of host haemoglobin and conversion of heme to hemozoin. Genetic knock-down approaches and detailed lipidomic studies confirmed that PfLPL1 protein plays key role in generation of neutral lipid stores in the parasite; neutral lipids are essentially required for hemozoin formation in the parasite, a vital function of the food-vacuole. Overall, this study identified specific role of PfLPL1 in the parasite which is essential for parasite survival.

microbiology↗

Wide sampling of natural diversity identifies novel molecular signatures of C4 photosynthesis

Introductory paragraphMuch of biology is associated with convergent traits, and it is challenging to determine the extent to which underlying molecular mechanisms are shared across phylogeny. By analyzing plants representing eighteen independent origins of C4 photosynthesis, we quantified the extent to which this convergent trait utilises identical molecular mechanisms. We demonstrate that biochemical changes that characterise C4 species are recovered by this process, and expand the paradigm by four metabolic pathways not previously associated with C4 photosynthesis. Furthermore, we show that expression of many genes that distinguish C3 and C4 species respond to low CO2, providing molecular evidence that reduction in atmospheric CO2 was a driver for C4 evolution. Thus the origin and architecture of complex traits can be derived from transcriptome comparisons across natural diversity.

plant biology↗