Biology subjects

Asthana, A.

Publications and source records attributed to Asthana, A..

11 recordsLinked to original sources

bioRxiv2025

Metabolic STAMP for deciphering GPCR-regulated insulin secretion by pancreatic β cells

Pancreatic islet {beta} cells integrate glucose and hormonal cues to control insulin secretion through spatially and temporally organized phosphorylation networks in health and diabetes. Here, using Metabolic STAMP (Synchronized Temporal-Spatial Analysis via Microscopy and Phosphoproteomics), we combine time-resolved phosphoproteomics, imaging, and kinase inhibition in mouse {beta} cells and human islets to map stimulus-specific GPCR signaling pathways. Metabolic STAMP reveals that GLP1-R and FFAR4 engage distinct, compartmentalized kinase programs, including GLP1-R-biased ERK activation, receptor-specific cAMP-PKA domains, and a phospho-ATAT1/HDAC6 node that differentially modulates microtubule acetylation and insulin secretion during GSIS based on stimulation conditions. These GPCR-responsive phospho-signatures and microtubule remodeling patterns are substantially conserved in human islets. Together, our data define an integrated, compartmentalized signaling architecture linking metabolic GPCR inputs, organelle remodeling, and insulin secretion, and provide a {beta}-cell phosphoproteomic resource that connects dynamic signaling nodes to human genetic risk and potential therapeutic targets.

physiology↗

bioRxiv2025

A novel approach for the extraction of nucleic acids using a hybrid paper-plastic device

Nucleic acid-based diagnostics play a crucial role in early and accurate disease detection. However, conventional extraction approaches are expensive, time-consuming, and require complex instrumentation and skilled personnel, which restricts their use in resource-limited settings. To develop and evaluate a simple, low-cost, and equipment-free paper-based microfluidic cassette device for rapid extraction of nucleic acids (DNA and RNA) from biological samples. A paper-plastic cassette was fabricated by laminating filter paper or glass fiber substrates between thermally bonded plastic sheets. The system was tested using Klebsiella pneumoniae (DNA) and HeLa cells (RNA) with different lysis buffer formulations. Extracted nucleic acids were quantified using a Qubit fluorimeter and assessed by gel electrophoresis and PCR amplification. Results were compared with standard commercial extraction kits. Among tested substrates, LF1 combined with lysis buffers 2, 4, and 5 yielded the highest quality DNA and RNA with 260/280 absorbance ratios of approximately 1.8 (DNA) and 2.0 (RNA). The nucleic acid yields were comparable to commercial kits, and the extracted material was suitable for direct downstream applications, including PCR. The developed paper-plastic cassette enables rapid, affordable, and equipment-free extraction of nucleic acids. This platform shows strong potential for point-of-care molecular diagnostics, particularly in low-resource and decentralized healthcare settings.

molecular biology↗

bioRxiv2025

Generation of uniform-sized spheroids using PDMS-based microwell devices as 3D cancer models for pharmacological profiling of drugs

Cancer researchers now consider spheroids a valuable in vitro model for cancer research and personalized medicine. They are used to studying cancer development, testing drug effectiveness, and potentially guiding treatment decisions for individual patients. Spheroids represent the simplest form of three-dimensional (3D) cellular arrangement and encapsulate the essential tumor microenvironment. These characteristics are crucial for studying processes such as tumor invasion, metastasis, angiogenesis, and cell cycle kinetics. In particular, spheroids excel in chemo-response assays where traditional monolayer cell cultures often fall short. A PDMS microwell device was developed to generate uniform-sized cancer spheroids. This device is user-friendly and capable of producing a large number of spheroids. The device measures 13 mm in diameter (1200 microwells per well if the device has microwells 400 m in size, and 300 microwells per well if the device has microwells 800 m in size). It advances 3D cultures by requiring only a small volume of cell culture supplements and is easy to manage. The hydrophobic nature of the PDMS device prevents cells from adhering to it, thereby promoting spheroid formation. Spheroids can be created on microwell devices, and subsequent experiments may either be conducted on the device or transferred to cell culture dishes for additional 3D biological assays. Seeding cells is notably easier compared to other 3D cell culture techniques, and the number of cells in each spheroid can be adjusted according to specific requirements. Overall, the PDMS-based microwell device offers a simple and efficient means to produce large quantities of uniform-sized spheroids for 3D cell culture studies, showcasing high throughput, short generation times, long-term effectiveness, and ease of handling.

bioengineering↗

bioRxiv2025

Dose Modeling for Mitochondrial Transplantation in Barrier-Limited Tissues

Mitochondrial transplantation is an emerging regenerative therapy aimed at preventing ischemia- reperfusion injury (IRI) by restoring oxidative metabolism in vulnerable tissues. While early clinical studies have demonstrated feasibility in pediatric myocardium and safety in adult stroke patients, dosing strategies remain undefined for barrier-limited organs where autoregulation, restricted perfusion, and anatomical constraints limit parenchymal uptake. We developed a physiologically grounded computational model to estimate route-specific input dose requirements for brain, spinal cord, and kidney. The model integrates organ-level parameters including tissue mass, regional perfusion or cerebrospinal flow, permeability-surface area product, and extraction efficiency. A benchmark dose of 2 x 106 mitochondria per gram, derived from validated cardiac protocols, was applied across delivery scenarios. Sensitivity analysis explored the effects of extraction fraction and tissue mass. Route-adjusted input requirements diverged quantitatively from cardiac-derived estimates, with up to 2-fold increases in central nervous system (CNS) targets and approximately 20% higher dosing required for the kidney. This framework provides a quantitative foundation for translational dose planning in IRI and related conditions.

pharmacology and toxicology↗

bioRxiv2025

Scalable 3D Bioprinting of Human Islets in a Pancreatic Decellularized Extracellular Matrix-Enriched Bioink for Beta-Cell Replacement Therapy

Allogeneic cell transplantation such as beta-cell replacement for treatment of type 1 diabetes (T1D) is constrained by poor graft survival and functionality, immune rejection, and the lack of scalable biomanufacturing processes. Here, we engineered functional human islet constructs that replicate the physiomimetic human pancreatic microenvironment by employing a clinically-scalable 3D bioprinting system. To support human islet viability and function, we developed alginate-based bioinks incorporating human pancreatic decellularized extracellular matrix (dECM). These bioink formulations were optimized for shear-thinning properties for extrusion of human islets, as well as selective permeability that supports nutrient and therapeutic molecule exchange. Extrusion-based printing parameters were refined to minimize shear stress-induced damage to human islets. The resulting bioprinted pancreatic constructs demonstrated robust structural integrity, high human islet viability (>85%), and long-term glucose-stimulated insulin secretion (GSIS) over a 21-days in vitro culture period, even at a high islet packing density (10,000 islet equivalent/mL) while free islet controls displayed a significant functional decline. The higher performance of bioprinted islets maybe attributable to the supportive 3D dECM-rich microenvironment mitigating culture-induced stress by recapitulating the islet pancreatic niche. This scalable 3D dECM-alginate bioprinted platform represents a new advanced functional material for advancing clinically translatable bio-artificial pancreas therapies for T1D.

bioengineering↗

bioRxiv2023

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since emerging in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a mutant recombinant SARS-CoV-2 (nsp15mut) expressing a catalytically inactive nsp15. Infection with SARS-CoV-2 nsp15 mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type (WT) virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI culture. SIGNIFICANCESevere acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a spectrum of respiratory disease ranging from asymptomatic infections to severe pneumonia and death. Innate immune responses during SARS-CoV-2 infection have been associated with clinical disease severity, with robust early interferon responses in the nasal epithelium reported to be protective. Thus, elucidating mechanisms through which SARS-CoV-2 induces and antagonizes host innate immune responses is crucial to understanding viral pathogenesis. CoVs encode various innate immune antagonists, including the conserved nonstructural protein 15 (nsp15) which contains an endoribonuclease (EndoU) domain. We demonstrate that SARS-CoV-2 EndoU is a crucial interferon antagonist, by providing further evidence for the role of the conserved CoV nsp15 in antagonizing innate immune activation, thereby optimizing CoV replication.

microbiology↗

bioRxiv2023

Initiation of a ZAKa-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L

RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2,5-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L inhibits viral infections. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells. Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAK. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38 are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes. Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAK kinase cascade leading to proinflammatory signaling and apoptosis. HighlightsO_LIRNase L signals antiviral innate immunity from the ribosome C_LIO_LIThe ribotoxic stress response requires the MAP3K ZAK C_LIO_LIRNA cleavage leads to transcription of proinflammatory genes C_LIO_LIZAK contributes to apoptosis downstream of RNase L activity C_LI

immunology↗

bioRxiv2022

Predicting regulators of epithelial cell state through regularized regression analysis of single cell multiomic sequencing

Chronic disease processes are marked by cell-specific transcriptomic and epigenomic changes. Single nucleus joint RNA- and ATAC-seq offers an opportunity to study the gene regulatory networks underpinning these changes in order to identify key regulatory drivers. We developed a regularized regression approach, RENIN, (Regulatory Network Inference) to construct genome-wide parametric gene regulatory networks using multiomic datasets. We generated a single nucleus multiomic dataset from seven adult human kidney biopsies and applied RENIN to study drivers of a failed injury response associated with kidney disease. We demonstrate that RENIN is highly effective tool at predicting key cis- and trans-regulatory elements.

genomics↗

bioRxiv2021

The MuvB Complex Binds and Stabilizes Nucleosomes Downstream of the Transcription Start Site of Cell-Cycle Dependent Genes

The chromatin architecture in promoters is thought to regulate gene expression, but it remains uncertain how most transcription factors (TFs) impact nucleosome position. The MuvB TF complex regulates cell-cycle dependent gene-expression and is critical for differentiation and proliferation during development and cancer. MuvB can both positively and negatively regulate expression, but the structure of MuvB and its biochemical function are poorly understood. Here we determine the overall architecture of MuvB assembly and the crystal structure of a subcomplex critical for MuvB function in gene repression. We find that the MuvB subunits LIN9 and LIN37 function as scaffolding proteins that arrange the other subunits LIN52, LIN54 and RBAP48 for TF, DNA, and histone binding, respectively. Biochemical and structural data demonstrate that MuvB binds nucleosomes through an interface that is distinct from LIN54-DNA consensus site recognition and that MuvB increases nucleosome occupancy in a reconstituted promoter. We find in arrested cells that MuvB primarily associates with a tightly positioned +1 nucleosome near the transcription start site (TSS) of MuvB-regulated genes. These results support a model that MuvB binds and stabilizes nucleosomes just downstream of the TSS on its target promoters to repress gene-expression.

molecular biology↗

bioRxiv2021

Specificity and Mechanism of Coronavirus, Rotavirus and Mammalian Two-Histidine-Phosphoesterases That Antagonize Antiviral Innate Immunity

2,5-oligoadenylate(2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2,5-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses and rotaviruses encode 2-5A degrading enzymes thereby antagonizing RNase L and its antiviral effects. A-kinase anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self double-stranded-RNAs that activate OAS. We show these enzymes, members of the two-histidine-phosphoesterase (2H-PE) superfamily, constitute a sub-family referred here as 2,5-PEs. 2,5-PEs from mouse coronavirus (CoV) MHV (NS2), MERS-CoV (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3,5-oligoribonucleotides, all cleaved 2,5-oligoadenylates efficiently, but with variable activity against other 2,5-oligonucleotides. The 2,5-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2,5-p3A3) producing mono- or di- adenylates with 2,3-cyclic phosphate termini. Our results suggest that elimination of 2-5A might be the sole function of viral 2,5-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCEViruses often encode accessory proteins that antagonize the host antiviral immune response. Here we probed the evolutionary relationships and biochemical activities of two-histidine-phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme, AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, that we refer to as 2,5-PEs, specifically degrade 2,5-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2,5- and not 3,5-phosphodiester bonds, producing cleavage products with cyclic 2,3-phosphate termini. Our study defines 2,5-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but which have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacologic strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.

microbiology↗

bioRxiv2020

Development of a Novel Class of Self-Assembling dsRNA Cancer Therapeutics: a Proof of Concept Investigation

Cancer has proven to be an extremely difficult challenge to treat. Several fundamental issues currently underlie cancer treatment including differentiating self from non-self, functional coupling of the recognition and therapeutic components of various therapies, and the propensity of cancerous cells to develop resistance to common treatment modalities via evolutionary pressure. Given these limitations, there is an increasing need to develop an all-encompassing therapeutic that can uniquely target malignant cells, decouple recognition from treatment, and overcome evolutionarily driven cancer resistance. We describe herein, a new class of programmable self-assembling dsRNA-based cancer therapeutics, that uniquely targets aberrant genetic sequences, and in a functionally decoupled manner, undergoes oncogenic RNA activated displacement (ORAD), initiating a therapeutic cascade that induces apoptosis and immune activation. As a proof-of-concept, we show that RNA strands targeting the EWS/Fli1 fusion gene in Ewing Sarcoma cells that are end-blocked with phosphorothioate bonds and additionally sealed with a 2-U modified DNA protector can be used to induce specific and potent killing of cells containing the target oncogenic sequence, but not wildtype.

bioengineering↗