Biology subjects

Cheema, A. K.

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

3 recordsLinked to original sources

bioRxiv2022

An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure

BackgroundUrinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical analysis methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. Methods3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment. ResultsSize-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories. ConclusionsWe demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork to develop future methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

cancer biology↗

bioRxiv2021

A multi-omics approach identifies pancreatic cancer cell extracellular vesicles as mediators of the unfolded protein response in normal pancreatic epithelial cells.

Although cancer-derived extracellular vesicles (cEVs) are thought to play a pivotal role in promoting cancer progression events, their precise effect on neighboring normal cells is unknown. In this study, we investigated the impact of pancreatic cancer ductal adenocarcinoma (PDAC) derived EVs on recipient non-tumorigenic pancreatic normal epithelial cells upon internalization. We show that PDAC cEVs increase the proliferation and invasive capability of treated normal cells. We further demonstrate that cEVs induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in treated normal pancreatic epithelial cells within 24 hours. Subsequently, these cells release several inflammatory cytokines. Leveraging a layered multi-omics approach, we analyzed EV cargo from a panel of 6 PDAC and 2 normal pancreas cell lines, using multiple EV isolation methods. We found that cEVs were enriched for an array of biomolecules which can induce or regulate ER stress and the UPR, including palmitic acid, sphingomyelins, metabolic regulators of tRNA charging and proteins which regulate trafficking and degradation. We further show that palmitic acid, at doses relevant to those found in cEVs, is sufficient to induce ER stress in normal pancreas cells. These results suggest that cEV cargo packaging may be designed to disseminate proliferative and invasive characteristics upon internalization by distant recipient normal cells, hitherto unreported. This study is among the first to highlight a major role for PDAC cEVs to induce stress in treated normal pancreas cells that may modulate a systemic response leading to altered phenotypes. For the first time, our study implicates cEV transported palmitic acid as a potential driver in this process. These findings highlight the importance of EVs in mediating disease etiology and open potential areas of investigation toward understanding the role of cEV lipids in promoting cell transformation in the surrounding microenvironment.

cancer biology↗

bioRxiv2018

Altered Fecal Microbiota and Urine Metabolome as Signatures of Soman Poisoning

The experimental pathophysiology of organophosphorus (OP) chemical exposure has been extensively reported. Here, we describe an altered fecal microbiota and urine metabolome that follows intoxication with soman, a lipophilic G class chemical warfare nerve agent. Non-anaesthetized Sprague-Dawley male rats were subcutaneously administered soman at 0.8 - 1.0 of the median lethal dose (LD50) and evaluated for signs of toxicity. Animals were stratified based on seizing activity to evaluate effects of soman exposure on fecal bacterial biota and urine metabolites. Soman exposure reshaped fecal bacterial biota by preferentially expanding Facklamia, Agrobacterium, Bilophila, Enterobacter, and Morganella genera of the Firmicutes and Proteobacteria phyla, some of which are known to hydrolyze OPs. However, analogous changes were not observed in the bacterial biota of the ileum, which remained the same irrespective of dose or seizing status of animals after exposure. Interestingly, when considering just the seizing status of animals, we found that the urine metabolome was markedly altered. Leukotriene C4, kynurenic acid, 5-hydroxyindoleacetic acid, norepinephrine, and aldosterone were excreted at much higher rates at 72 hrs in seizing animals, consistent with early multi-organ involvement during soman poisoning. However, at 75 days post soman exposure, bacterial biota stabilized and no differences were observed. These findings demonstrate the feasibility of using the dysbiosis of fecal bacterial biota in combination with urine metabolome alterations as forensic evidence for OP exposure temporally.\n\nImportanceThe paucity of assays to determine physiologically relevant OP exposure presents an opportunity to explore the use bacterial sentinels in combination with urine to assess changes in the exposed host. Recent advances in technologies and computational approaches have enabled researches to survey large community level changes of gut bacterial biota and metabolomic changes in various biospecimens. Here, we profile combined changes in bacterial biota and urine metabolome due to chemical warfare OP exposure. The significance of our work is to reveal that monitoring bacterial biota and urine metabolites as surrogates of OP exposure in biospecimens suitable for existing clinical laboratory workflows is plausible without the need for the development of new technology, invasive procedures, or complicated analytical approaches. The larger value of such an approach is that any setting with a moderate clinical chemistry and microbiology capability can determine pre-symptomatic exposure to enhance current triage standards in case of mass exposures, refugee movements, humanitarian missions, and training settings once an algorithm has been validated. In the event of \"potential\" exposures by time or distance, this assay can be further developed to estimate affected radius or time dimension for health monitoring and treatment interventions.

physiology↗