Biology subjects

Challa, A. K.

Publications and source records attributed to Challa, A. K..

6 recordsLinked to original sources

bioRxiv2026

ER Stress-Induced beta-Cell Apoptosis is Linked to Novel Select Lipid Signaling at the Transcriptional Level: Implications in T1D Development

Type 1 diabetes (T1D) is a consequence of {beta}-cell death. ER stress precedes T1D onset and prolonged ER stress in {beta}-cells can lead to {beta}-cell apoptosis. We reported that lipid signaling generated by the Ca2+-independent phospholipase A2{beta} (iPLA2{beta}), encoded by Pla2g6, participates in ER stress-mediated {beta}-cell apoptosis. {beta}-Cell membranes are enriched in arachidonic acid containing glycerophospholipids and the iPLA2{beta} catalyzes the hydrolysis of arachidonic acid in ER stressed {beta}-cells. Metabolism of arachidonic acid leads to the generation of various proinflammatory lipids, raising the possibility that they contribute to ER stress and {beta}-cell death leading to T1D. However, molecular mechanisms by which such {beta}-cell-iPLA2{beta}-derived lipid (iDL) signaling contributes to {beta}-cell apoptosis are not understood. It is well known that ER stress-mediated {beta}-cell apoptosis is associated with induction of transcription factors, NF{kappa}B and STAT1. We report here that both induce Pla2g6 and, unexpectedly, we find that iPLA2{beta}, which lacks DNA-binding motifs, associates with NFkB, Stat1, and Pla2g6 promoter regions. Consistently, p65-NF{kappa}B and pSTAT1 induction is reduced with select inhibition or knockdown of iPLA2{beta}. Surprisingly, iPLA2{beta} expression is also reduced by select inhibition of iPLA2{beta}, raising the possibility of feedback regulation by iDLs. In support, we find that select iDLs, recognized to be proinflammatory, enhance association of iPLA2{beta} with Pla2g6, Nfkb, and Stat1 promoter regions leading to induction of all three gene products and {beta}-cell apoptosis. Our findings reveal previously unrecognized transcriptional regulation by iDL signaling and, iPLA2{beta} itself, that leads to gene products that promote {beta}-cell apoptosis. Analogous findings in human islets validate this mechanism raising the possibility that targeting select lipid signaling can reduce ER stress in {beta}-cells and ameliorate T1D development.

cell biology↗

bioRxiv2021

Defining function of wild-type and patient specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss or gain of function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding TP53 effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet TP53 mutations when present in tumors are associated with poor prognosis. Employing a kRASG12D-driven ERMS tumor model and newly generated tp53 null (tp53-/-) zebrafish, we define both wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumor initiation, where tp53 loss expands tumors initiation from <35% to >97% of animals. Next, characterizing three patient-specific mutants finds that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, while the TP53P153{Delta} and TP53Y220C mutants define two structural mutations that predispose to head musculature ERMS.

cancer biology↗

bioRxiv2020

Transcriptional regulation of structural and functional adaptations in a developing adulthood myocardium

The development of the heart follows a synergic action of several signaling pathways during gestational, pre- & postnatal stages. The current study aimed to investigate whether the myocardium experiences transcriptional changes during the transition from post-natal to adult hood stages. Herein, we used C57/Bl6/J mice at 4 (28-days; post-natal/PN) and 20 weeks (adulthood/AH) of ages and employed the next generation RNAseq (NGS) to profile the transcriptome and echocardiography analysis to monitor the structural/functional changes in the heart. NGS-based RNA-seq revealed that 1215 genes were significantly upregulated and 2549 were down regulated in the AH versus PN hearts, indicating a significant transcriptional change during this transition. A synchronized cardiac transcriptional regulation through cell cycle, growth hormones, redox homeostasis and metabolic pathways was noticed in both PN and AH hearts. Echocardiography reveals significant structural and functional (i.e. systolic/diastolic) changes during the transition of PN to adult stage. Particularly, a progressive decline in ejection fraction (EF) and cardiac output was observed in AH hearts. These structural adaptations are in line with critical signaling pathways that drive the maturation of heart during AH. Overall, we have presented a comprehensive transcriptomic analysis along with structural-functional relationship during the myocardial development in adult mice.

genomics↗

bioRxiv2020

Physiological and Metabolic Features of Mice with CRISPR/Cas9-Mediated Loss-of-Function in Growth Hormone-Releasing Hormone

Our previous study demonstrated that the loss of growth hormone releasing hormone (GHRH) results in increased lifespan and improved metabolic homeostasis in the mouse model generated by classical embryonic stem cell based gene-targeting method. In this study, we targeted the GHRH gene using the CRISPR/Cas9 technology to avoid passenger alleles/mutations and performed in-depth physiological and metabolic characterization. In agreement with our previous observation, male and female GHRH-/- mice have significantly reduced body weight and enhanced insulin sensitivity when compared to wild type littermates. Dual-energy X-ray absorptiometry showed that there were significant decreases in lean mass, bone mineral content and density, and a dramatic increase in fat mass of GHRH-/- mice when compared to wild type littermates. Indirect calorimetry measurements including oxygen consumption, carbon dioxide production and energy expenditure were dramatically reduced in GHRH-/- mice compared to wild type mice. Respiratory exchange ratio was significantly lower in GHRH-/- mice during the light cycle, but not during the dark cycle, indicating a circadian related metabolic shift towards fat utilization in the growth hormone deficient mice. The novel CRISPR/Cas9 GHRH-/- mice are exhibiting the consistent and unique physiological and metabolic characteristics, which might mediate the longevity effects of growth hormone deficiency in mice.

physiology↗

bioRxiv2018

Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene induced by CRISPR-Cas9 RNPs

Nodal-related protein (ndr2) is a member of the transforming growth factor type {beta} superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development, and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced, and the remaining alleles were obtained via random insertion, gene targeting (TALEN), or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos. We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted PCR amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain- specific mutations and provides further insights into the structure-function of the ndr2 gene.

genetics↗

bioRxiv2018

NF1 deficiency correlates with estrogen receptor signaling and diminished survival in breast cancer

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. Much of our understanding of the mechanisms underlying the functional loss of NF1 comes from mouse models that do not completely recapitulate the phenotypes of human NF1. We utilized CRISPR-Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor and progesterone receptor. We identified distinct Nf1 isoforms that were altered during tumorigenesis.\n\nTo evaluate NF1 in human breast cancer, we analyzed genomic changes in a breast cancer dataset of 2,000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. These results demonstrated the significant role NF1 plays in both NF1-related breast cancer and sporadic breast cancer.

cancer biology↗