Biology subjects

Upadhyay, P.

Publications and source records attributed to Upadhyay, P..

10 recordsLinked to original sources

bioRxiv2026

Dynamic s-acylation of schizophrenia-linked antioxidants explicate the redox flexibility of human microglia during inflammation.

Cysteine proteome essentially regulates cellular adaptability during stress. Proteomic cysteines are reversibly modified to avoid loss through oxidation. Reversible cysteine-modifications beneficially preserve cellular stress-response mechanisms by regulating the transient activation of substrate proteins. Cysteine acylation (or s-acylation) is one such extensively documented reversible protein modification. Inflammation-responsive changes in s-acylation facilitate the plasticity of immune-partitioned brains by dynamically determining the utilization of stress-buffering (substrate) cysteine-proteins in microglia, the primary immune-reactive brain cells. However, the inflammation-responsiveness of cysteine-acylation in microglia is not elaborately measured at a large-scale. Therefore, we observed the differential (quantitative) s-acylation of proteins in cultured human (HMC3) microglia by implementing comparative MLCC-based chemoproteomic workflows. Parallelly, we characterized the molecular consequences of HMC3 cell treatment with 2-bromopalmitate, a general s-acylation inhibitor, by performing immunocytochemistry and proteomic experiments. We also identified the s-acylation-dependent microglial inflammatory responses through live spatiotemporal tracking and confocal microscopy, following treatment of microglia with PAMPs (LPS or IFNy). Finally, from our proteomic studies of postmortem human brain tissues, we confirmed major protein-causals of schizophrenia, a redox-related brain disorder, to undergo inflammation-responsive s-acylation. This report provides a first comprehensive library of cysteine-proteins differentially processed through inflammation-responsive acylation in microglia. It also outlines the inflammation-responsive s-acylation of key NRF2-antioxidants, including peroxiredoxins (especially PRDX2) and glutathione synthetase (GSS), as a biochemical signature of early-stage inflammation and oxidative-stress in microglia. More importantly, the innovative proteo-informatic workflows designed for this research work can be reliably repurposed to identify bifunctional theragnostic protein markers of many complex disorders and diseases.

biochemistry↗

bioRxiv2026

Adaptive Landscapes of Plasmodium Falciparum Dihydrofolate Reductase Reveal Pathways to Antifolate Resistance

Antifolate resistance in Plasmodium falciparum dihydrofolate reductase (pfDHFR) remains a major challenge for malaria control. To understand how this enzyme maintains function under antifolate selection, we developed PfPATH, a computational framework that integrates mutational fitness measurements, evolutionary interactions, and structural information to map adaptive trajectories. PfPATH adaptive walks reveal that evolution is constrained to a narrow ridge in sequence space defined by a small subset of residues. Most resistance mutations are individually deleterious and become viable only when supported by stabilizing changes, restricting adaptation to a few high-resistance pathways. Structure network analyses show that these mutations do not disrupt the global enzyme architecture but instead reorganize internal communication to preserve catalytic function. Consistent with this, molecular dynamics and free-energy analyses indicate that resistant variants remain stable while sampling a broader ensemble of low-energy conformational substates. Together, these results reveal a narrow and predictable route to antifolate escape.

biophysics↗

bioRxiv2025

Monoclonal antibody to Filarial serpin Wb123 impedes the urokinase plasminogen activator receptor-mediated Alternative activation of macrophages

Macrophages play a multifaceted role in mounting an immune response against pathogens through phagocytosis, antigen presentation and cytokine production. Activated macrophages are critical for the initiation, maintenance, and resolution of inflammation. They can either be classically activated (Th1 response), which promotes inflammation or alternatively activated (Th2 response), which resolves inflammation. The alternate activation of macrophages, along with diminished T cell response, is thought to be an important immune evasion strategy employed by the filarial parasites. However, the mechanism behind filaria-induced alternate activation remains unclear. In this study, by using in-silico approach we identified filarial serpins which are highly expressed in the infective L3 larval stages. We characterized the filarial serine protease inhibitor, Wb123 and established its role in alternate activation. We observed that Wb123 impairs nitric oxide (NO) and reactive oxygen species (ROS) expression, which plays a crucial role in mounting a strong immune response. Surprisingly, we also observed elevated IL6 expression and phosphorylation of STAT3, indicating IL-6-dependent alternate activation. Our findings indicate that the urokinase plasminogen activator receptor (uPAR) is essential for Wb123-induced alternate activation. Furthermore, we demonstrated that the monoclonal antibody MAbG8 impedes the Wb123-induced alternate activation, as shown by reduced CD163 expression along with increased ROS and uPA expression in response to lipopolysaccharide (LPS) and interferon-gamma (IFN-{gamma}). Overall, our study identifies the filarial serpin Wb123 and highlights its potent role in alternative activation. This interaction reveals, for the first time a mechanism exploited by filarial parasites to evade a strong pro-inflammatory immune response.

cell biology↗

bioRxiv2025

Wearable Photonic Device for Multiple Biomarker Sampling and Detection Without Blood Draws

Blood draws or phlebotomy using needles have been practiced in clinics for several centuries and it often causes pain, discomfort, and inconvenience. Herein, we create a wearable photonic device by integrating a microlens array (MLA) and an optic microneedle array (OMNA) functionalized with immunobinding capacity for safe and needle-free biomarker sampling and detection. The MLA-integrated OMNA amplified and transmitted LED light at 595 nm into the skin through the OMNA, bypassing the light-absorbing melanin in the epidermal layer, and evenly distributing it in the capillary-enriched dermis. The 595 nm light was preferably absorbed by hemoglobin (Hb) and oxygen-Hb within the capillaries, triggering thermal dilation of capillaries without damaging them or causing petechiae. The light illumination resulted in remarkable increases in the concentrations of various blood biomarkers in the skin owing to capillary dilation and biomarker extravasation. These extravasated biomarkers bound specifically to the OMNA, which was covalently decorated with capture antibodies, each microneedle immobilized with one specific antibody. The multi-functional OMNA was extensively modified to amplify the immunobinding signals and achieve sensitivity superior to that of enzyme-linked immunosorbent assay (ELISA) kits. As a proof of concept, we validated the functionality of the prototype for minimally invasive sampling and precise multiplexed blood biomarker detection in two mouse models to quantify acute inflammation and specific antibody production. This cost-efficient device provides a promising platform for blood-free, multiplexed detection of blood biomarkers.

bioengineering↗

bioRxiv2024

Enhancing NADPH to Restore Redox Homeostasis and Lysosomal Function in G6PD-Deficient Microglia

Microglia, the residential immune cells in the central nervous system (CNS) exhibited in multiple states from resting to activated, play a significant role in neurogenesis, myelination, synaptic transmission, immune surveillance, and neuroinflammation. The aggravated inflammatory response by microglia triggers the generation of superoxide, which often causes the degeneration of neurons, leading to the development of Parkinsons and Alzheimers. The oxidative stress is key to many neurological disorders, often regulated by many genes. The terminal neutralization of oxidative stress is mediated by NADPH and glutathione. The cytosolic NADPH level is majorly contributed by a key enzyme called glucose-6-phosphate dehydrogenase (G6PD). The deficiency of G6PD is associated with hemolytic anemia, diabetes, cardiovascular, autoimmune, and neurological disorders. Our recent study indicated that G6PD deficiency decreases cytosolic NADPH levels and alters redox homeostasis and lysosomal function in microglia. Therefore, replenishment of NADPH is crucial for targeting G6PD deficiency-mediated microglial dysfunctions. This research promotes alternate metabolic pathways by targeting the expression and activity of enzymes such as isocitrate dehydrogenase 1 (IDH1) and malic enzyme 1 (ME1), which are responsible for cytoplasmic NADPH production. Metabolites like citric and malic acid supplementation promote NADPH production and reduce microglial oxidative stress. Additionally, using another group of small-molecule metabolites, such as dieckol and resveratrol, enhances the expression of IDH1 and ME1 enzymes to resolve potential tissue heterogeneity. Finally, combining these metabolites supplementation increased NADPH production and restored redox homeostasis and lysosomal function in G6PD deficient microglia, indicating their further use as potential therapeutics against G6PD deficiency disorders. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/607918v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@5e69f2org.highwire.dtl.DTLVardef@7d9015org.highwire.dtl.DTLVardef@17e5500org.highwire.dtl.DTLVardef@fe9bc5_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

G6PD deficiency mediated impairment of iNOS and lysosomal acidification affecting phagocytotic clearance in microglia in response to SARS-CoV-2

The glucose-6-phosphate dehydrogenase (G6PD) deficiency is X-linked and is the most common enzymatic deficiency disorder globally. It is a crucial enzyme for the pentose phosphate pathway and produces NADPH, which plays a vital role in the regulation of oxidative stress of many cell types. The deficiency of G6PD causes hemolytic anemia, diabetes, cardiovascular and neurological disorders. Notably, the patient with G6PD deficiency was severely affected by SARS-CoV-2 and showed prolonged COVID-19 symptoms, neurological impacts, and high mortality. However, the mechanism of COVID-19 severity in G6PD deficient patients is still ambiguous. Here, using a CRISPR-edited G6PD deficient human microglia cell culture model, we observed a significant reduction in NADPH and an increase in basal reactive oxygen species (ROS) in microglia. Interestingly, the deficiency of the G6PD-NAPDH axis impairs induced nitric oxide synthase (iNOS) mediated nitric oxide (NO) production which plays a fundamental role in inhibiting viral replication. Surprisingly, we also observed that the deficiency of the G6PD-NADPH axis reduced lysosomal acidification, which further abrogates the lysosomal clearance of viral particles. Thus, impairment of NO production and lysosomal acidification as well as redox dysregulation in G6PD deficient microglia altered innate immune response, promoting the severity of SARS-CoV-2 pathogenesis.

cell biology↗

bioRxiv2022

A mechanistic insight into Nigella sativa mediated anticancer effect on breast cancer through regulation of various miRNAs: An in vitro & in vivo study

BackgroundCancer continues to threat the mortal alongside scientific community with its burgeoning grasp. Although efforts have been directed to tame cancer by radiotherapy, photodynamic therapy, chemotherapy it came at a cost of fatal side effects. PurposePlant derived bioactive compounds carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. This study analyzed anti-cancerous potential of volatile oil, extracted from Nigella sativa, in-vitro against MDA-MB-231, MCF-7 & in-vivo on tumor growth in mice after successful oral administration. Study DesignOur strategic approach employed solvent extraction of black seed oil (BSO) to highlight orchestrated use of its potent integrants - TQ, Carvacrol & TA which in modest amounts show anti-cancerous properties compared to their individual treatment. We attempted to show this cost effective, safe & bioavailable form of dealing with the atrocities of breast cancer by means of MTT, Apoptotic, Western Blot Assays besides Transwell & Wound healing Assay. Reduction in the solid tumour in-vivo & near normalcy restoration of tissue section architecture from the BSO treated tumour sets are indicative of the better anti-tumorigenic potential of BSO. MethodsBSOs Solvent extraction was performed followed by its characterization. MTT aided cytotoxicity study of BSO alongside major components in PBMC & cancer cells while its efficacy was presented by flow cytometric ROS analysis, cell cycle arrest & apoptosis assessment. Anti-migratory potential evaluated by Wound Healing, Transwell Migration & Western Blot while the expression study of a wide range of proteins, miRNAs & the in-vivo studies undertaken climaxed the confirmation of the anti-cancerous potential of BSO. ResultsComparatively reduced concentrations of TQ, TA & Carvacrol in BSO played a synergistic role to enhance apoptotic potential via Caspase 7 & 9, through enhanced ROS & expression of apoptotic family of proteins, miRNAs besides uplifting the anti-migratory perspective by effectively enhancing E-cad & downregulating lamellipodia, filopodia assembly & MMPs in MCF-7 & MDA-MB-231. Similar observations in-vivo outlined the therapeutic potential of BSO. ConclusionThis study culminates isolation & processing of BSO in a simplified procedure, thereby aiming at a more lucrative paradigm to be accepted in contemporary phytomedicine research.

cancer biology↗

bioRxiv2022

Preclinical efficacy of bacosides-lauric acid nano-herbal formulation incomparison to rivastigmine for treatment of Alzheimer's disease

Despite decades of rigorous scientific endeavors for Alzheimers disease (AD) drug development, massive failures in clinical trials is continuously posing healthcare and societal burden. Currently recommended single targeted drugs including rivastigmine with limited bioavailability can alleviate AD symptoms only for a limited period of time but unable to reverse the disease progression. Recent evidences consider poly-pharmacological and multi-targeted agents aiming at amyloid and tau burden, neuroinflammation, neuroprotection and cognitive enhancement as potential treatment options. In this regard, bioactive herbal compounds with holistic action and minimal adversities have gained prominence, though lacunae in their scientific validation and limited bio-permeability to cross BBB represent major hurdles. Previously, we showed that lactoferrin conjugated PEG-S-S-PLA-PCL-OH efficaciously delivered herbal compounds to brain and such nano-herbal formulation of bacosides-lauric acid (BAN-LAN) attenuated neuronal damages induced by scopolamine in vitro. Here, we tested the preclinical potential of BAN-LAN in reversal of AD pathologies in 5XFAD transgenic mice. Our nano-herbal formulation substantially reduced amyloid burden by clearing A{beta} plaques in the hippocampus of 5XFAD mice and also attenuated a{beta}42 induced alterations in AD associated gene expression in hippocampal neurons in vitro. It showed neuroprotection by rescuing neuronal damage and promoting neurogenesis in the hippocampus of AD transgenic mice. Anti-neuroinflammatory properties were also exhibited by the formulation as evident from inhibition of hippocampal astrocytic and microglial activation in 5XFAD mice. BAN-LAN showed cognitive efficacy by restoring memory impairment in AD transgenic mice and effects were more pronounced than unconjugated natural form and rivastigmine. These findings suggest that BAN-LAN may serve as a promising therapeutic agent for AD with better brain penetration and targeting multiple AD pathways.

pharmacology and toxicology↗

bioRxiv2020

Diversified agroforestry systems improve carbon footprint and farmers livelihood under limited irrigation conditions

Increasing weather aberrations cause frequent crop failure in monoculture cropping system. Specialized crop production systems, where few seasonal crops occupy vast arable lands, resulting in more biotic and abiotic stresses in agri-ecosystem. Therefore a diversified agroforestry systemwas evaluated to ensure resilience underlimited water conditions, with an aim to augment carbon footprint with enhanced productivity and profitability. The study hypothesised that integration of perennial fruits trees with seasonal crops will have benign effect for sequestering more carbon and improving livelihood of the farmers. This is one of the first timesthat arid fruits tress along with leguminous,and other low water requiring crops were studied for improved carbon sequestration, livelihood of the farmers andfor better resilience in production system. The experimental findings showed that arid fruit trees along with leguminous, oilseeds and cash crops resulted in higher profitability and thus improved livelihood of the farmersin arid and semi-arid areas of South Asia. Diversified phalsa-mung bean-potato and moringa-mung bean-potato were the most productive agroforestry system (36.7t/ha and 36.2 t/ha respectively. Under limited irrigation conditions, Karonda (Carisa spp.)-mung bean potato system was found best in improving livelihood with maximum net return of $ 3529.1/ha with higher profitability/day ($ 19.9/day). Phalsa -MB-potato system was also recorded maximum water use efficiency (33.0 kg/ha-mm), whereas density of SOC was in Phalsa-cowpea-mustard (9.10 Mg/ha) and moringa-mung bean -potato AFS (9.16 Mg/ha). Carbon footprint analysis revealed that maximum net C gain was in Phalsa-mung bean -potato system (7030 Carbon equivalent kg CE/ha/year).

ecology↗

bioRxiv2018

Activation of Multiple Signalling Pathways by P152Lp53 Mutant Reveals New Gain-of-function Implicating Tumorigenesis

TP53 is the most frequently mutated tumor suppressor gene in context of all varieties of cancer, yet biochemical characterization of several reported mutations with probable biological significance have not been accomplished. We have identified a relatively rare proline to leucine mutation (P152L) in p53 in an Indian oral cancer patient sample at the fade end of its DNA binding domain (DBD). Although P152Lp53 DBD potentially binds to DNA, the full length protein is completely devoid of DNA binding ability at its cognate site. Interestingly, P152Lp53 can efficiently tetramerize. Significantly, this mutant when expressed in p53 null cell line, was found to induce cell mobility, proliferation, and invasion as compared to vector control. Also, enhanced tumorigenic potential was observed when cells expressing P152Lp53 were xenografted into nude mice, the mechanistic details of which were also investigated upon where several of the pathways were found to be upregulated such as Cell-Cell/Cell-ECM signalling, EGFR signalling, Rho-GTPase signalling. Taken together, this study establishes P152Lp53 as a new gain of function mutant.

cancer biology↗