Biology subjects

Shah, A. P.

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

9 recordsLinked to original sources

bioRxiv2026

Hierarchical control of remyelination by heparan sulfate sulfation

Efficient myelin regeneration requires coordinated signaling between oligodendrocyte progenitor cells (OPCs) and the extracellular matrix, yet how specific heparan sulfate sulfation patterns regulate this process remains poorly understood. The extracellular endosulfatases Sulf1 and Sulf2 selectively remove 6-O-sulfate groups from heparan sulfate proteoglycans (HSPGs). We previously demonstrated that these enzymes are highly expressed by OPCs following demyelination and inhibit OPC recruitment, differentiation, and remyelination through activation of BMP and WNT signaling. We investigated whether sulfotransferases that establish 2-O and 6-O sulfation during HSPG biosynthesis regulate remyelination. Using OPC-specific conditional knockout models, deletion of the 2-O sulfotransferase Hs2st1 enhanced OPC recruitment, oligodendrocyte differentiation, and remyelination through suppression of BMP and WNT signaling. Loss of 2-O sulfation induced compensatory remodeling of the heparan sulfate glycome, with a marked enrichment of 6-O-sulfated HS species. Conversely, deletion of 6-O sulfotransferases impaired OPC recruitment and differentiation and reversed the Hs2st1 phenotype, identifying 6-O sulfation as the dominant functional determinant of remyelination. Together, these findings establish hierarchical control of remyelination by heparan sulfate sulfation, demonstrating that compensatory remodeling of the heparan sulfate glycome governs regenerative signaling following demyelination.

neuroscience↗

bioRxiv2024

Engineering inducible signaling receptors to enable erythropoietin-free erythropoiesis

Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible EPO receptors (iEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of iEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media--EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how synthetic biology and genome editing may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated cell engineering strategies.

synthetic biology↗

bioRxiv2023

IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events

The metazoan-specific Integrator complex catalyzes 3 end processing of small nuclear RNAs (snRNAs) as well as premature transcription termination events that attenuate expression of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are always required for complex function. Here, we show that IntS6 (Integrator subunit 6) over-expression is sufficient to block Integrator function at a subset of Drosophila protein-coding genes, while having no effect on snRNA processing or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits from the rest of the Integrator complex and thus the only loci affected are those where the phosphatase module is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not associate with Integrator, are also sufficient to inhibit Integrator function at select loci. Altogether, these results show that the phosphatase module is critical and limiting at only a subset of Integrator regulated genes and point to recruitment of PP2A via IntS6 as a tunable step that can be used to modulate transcription termination efficiency.

molecular biology↗

bioRxiv2023

DNA metabarcoding using new rbcL and ITS2 metabarcodes collectively enhance detection efficiency of medicinal plants in single and polyherbal formulations

With the widespread adoption of barcoding and next-generation sequencing, metabarcoding is emerging as a potential tool for detecting labelled and unlabelled plant species in herbal products. In this study, newly designed rbcL and ITS2 metabarcode primers were validated for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single drugs and 15 polyherbal formulations procured from the Indian market. The rbcL metabarcode demonstrated detection efficiencies of 86.7% and 71.7% in gDNA plant pools and biomass mock controls, respectively, while the ITS2 metabarcode demonstrated 82.2% and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative detection efficiency increased by 100% and 90%, respectively. A total of 79% cumulative detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted plant species present within mock controls as well as in herbal formulations. Our results demonstrated the applicability of multilocus strategies in metabarcoding as a potential tool for detecting labelled and unlabelled plant species in herbal formulations.

molecular biology↗

bioRxiv2022

Comprehensive analysis using DNA metabarcoding, PCR, and HPLC unveils the adulteration in Brahmi herbal products

BackgroundThe herbal products market is expanding and creating a bottleneck for raw materials. Hence, economically motivated adulteration has a high prevalence. DNA barcoding and species-specific PCR assays are now revolutionising the molecular identification of herbal products and are included in a number of pharmacopoeias for the identification of raw materials. High-throughput sequencing with barcoding advances toward metabarcoding, which enables the identification of unintentionally or intentionally unlabelled plant material present in herbal products. Brahmi is one of the most commercially significant and nootropic botanicals, with great controversy over the terms "Brahmi" being used to describe both Bacopa monneri (BM) and Centella asiatica (CA) species. PurposeThis study evaluates DNA-based methods for Brahmi herbal products with the traditional HPLC-based analytical approach in order to assess their effectiveness. MethodsWe employed a species-specific PCR assay, DNA metabarcoding using rbcL minibarcode, and HPLC to detect the presence of the Brahmi (either BM or CA) in eighteen market samples. All the methods have been validated using in-house blended formulations. ResultsComprehensive analysis of all three methods revealed the presence of 22.2%, 55.6%, and 50.0% of Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively, in Brahmi market formulations, whereas blended formulations only exhibited targeted plant species with all three methods. ConclusionSpecies-specific PCR can be used as a cost-effective and rapid method to detect the presence of the Brahmi, while in high-throughput methods, DNA metabarcoding can be used to detect the presence of widespread adulterated botanicals, and further, bioactive compounds could be detected by HPLC. These results emphasise the need for quality control of the marketed Brahmi herbal products as well as the implementation of all methodologies in accordance with fit for purpose.

molecular biology↗

bioRxiv2021

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. Summary StatementIn this work, we developed and thoroughly characterized several unique models of hepatoblastoma derived from the HepT1 cell line that show high-risk features.

cancer biology↗

bioRxiv2020

Constitutive deletion of translin (Tsn) enhances locomotor response to amphetamine: role of developmental effects

The translin/trax microRNA-degrading enzyme mediates activity-induced changes in translation that underlie several long-lasting forms of cellular plasticity. As translin and trax are expressed in dopaminergic and striatal neurons, we proceeded to investigate whether deletion of Tsn blocks amphetamine sensitization, a long-lasting, translation-dependent form of behavioral plasticity, Although we expected constitutive Tsn deletion to impair amphetamine sensitization, we found, instead, that it enhances the hyperlocomotion produced by the initial dose of amphetamine. Since these mice display elevated adiposity, which alters pharmacokinetics of many drugs, we measured brain amphetamine levels in Tsn knockout mice and found that these are elevated. We also found that diet-induced increases in adiposity in WT mice correlate with elevated brain amphetamine levels. As amphetamine and its analogues are widely used to treat attention deficit disorder, which is associated with obesity, further studies are needed to assess the impact of adiposity on amphetamine levels in these patients.

neuroscience↗

bioRxiv2020

Genetic inactivation of the translin/trax microRNA-degrading enzyme phenocopies the robust adiposity induced by Translin (Tsn) deletion

ObjectiveDeletion of Translin (Tsn) from mice induces an unusual metabolic profile characterized by robust adiposity, normal body weight and glucose tolerance. Translin (TN) protein and its partner, trax (TX), form the TN/TX microRNA-degrading enzyme. Since the microRNA system plays a prominent role in regulating metabolism, we reasoned that the metabolic profile displayed by Tsn KO mice might reflect dysregulation of microRNA signaling. MethodsTo test this hypothesis, we inserted a mutation, E126A, in Tsnax, the gene encoding TX, that abolishes the microRNA-degrading enzymatic activity of the TN/TX complex. In addition, to help define the cell types that drive the adiposity phenotype, we have also generated mice with floxed alleles of Tsn or Tsnax. ResultsIntroduction of the E126A mutation in Tsnax does not impair expression of TN or TX proteins or their co-precipitation. Furthermore, these mice display selective increases in microRNAs that match those induced by Tsn deletion, confirming that this mutation in Tsnax inactivates the microRNA-degrading activity of the TN/TX complex. Mice homozygous for the Tsnax (E126A) mutation display a metabolic profile that closely mimics that of Tsn KO mice. Selective deletion of Tsn or Tsnax from either adipocytes or hepatocytes, two candidate cell types, does not phenocopy the elevated adiposity displayed by mice with constitutive Tsn deletion or the Tsnax(E126A) mutation. Furthermore, global, conditional deletion of Tsn in adulthood does not elicit increased adiposity. ConclusionTaken together, these findings indicate that inactivation of the TN/TX microRNA-degrading enzyme during development is necessary to drive the robust adiposity displayed by Tsn KO mice.

cell biology↗

bioRxiv2018

Monocyte and Macrophage Subtypes as Paired Cell Biomarkers for Coronary Artery Disease

Background: Monocytes and macrophages are central to atherosclerosis, but how they mark progression of human coronary artery disease (CAD) is unclear. We tested whether patients monocyte subtypes paired with their derived macrophage profiles correlate with extent of CAD.\n\nMethods: Peripheral blood was collected from 30 patients undergoing cardiac catheterization, and patients were categorized as having no significant CAD, single vessel disease, or multivessel disease according to the number of affected coronary arteries. Mononuclear cells were measured for monocyte markers CD14 and CD16 by flow cytometry, and separate monocytes were cultured into macrophages over 7 days and measured for polarization markers CD86 and CD206.\n\nResults: At baseline, patients with greater CAD burden were older with higher rates of statin use, whereas all other characteristics were similar across the spectrum of coronary disease. Non-classical (CD14loCD16hi) and all CD16+ monocytes were elevated in patients with single vessel and multivessel disease compared to those without significant CAD (8.6% and 10.5% vs. 2.8%, p < 0.05), whereas regulatory M2 macrophages (CD206+) were decreased in patients with single vessel and multivessel disease (0.34% and 0.34% vs. 1.4%, p < 0.05). An inverse relationship between paired CD16+ monocytes and M2 macrophages marked CAD severity. CAD was also found to be more tightly associated with CD16+ cells than age or traditional cardiovascular risk factors on multiple regression analysis of these patients.\n\nConclusions: CAD extent is correlated directly with CD16+ monocytes and inversely with M2 (CD206+) macrophages, suggesting circulating monocytes may influence downstream polarization of lesional macrophages. These measures of monocyte and macrophage subtypes hold potential as biomarkers in CAD.

physiology↗