Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients.

BACKGROUND: Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS: UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS: Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS: Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.

Prevalence and Impact of Preexisting Comorbidities on Overall Clinical Outcomes of Hospitalized COVID-19 Patients.

COVID-19 risk increases with comorbidities, and the effect is magnified due to the contribution of individual and combined comorbidities to the overall clinical outcomes. We aimed to explore the influence of demographic factors, clinical manifestations, and underlying comorbidities on mortality, severity, and hospital stay in COVID-19 patients. Therefore, retrospective chart reviews were performed to identify all laboratory-confirmed cases of SARS-CoV-2 infection in Apollo Hospitals, Hyderabad, between March 2020 and August 2020.A total of 369 confirmed SARS-CoV-2 cases were identified: 272 (73.7%) patients were male, and 97 (26.2%) were female. Of the confirmed cases, 218 (59.1%) had comorbidities, and 151 (40.9%) were devoid of comorbidities. This study showed that old age and underlying comorbidities significantly increase mortality, hospital stay, and severity due to COVID-19 infection. The presence of all four comorbidities, diabetes mellitus (DM) + Hypertension (HTN) + coronary artery disease (CAD) + chronic kidney disease (CKD), conferred the most severity (81%). The highest mortality (OR: 44.03, 95% CI: 8.64-224.27) was observed during the hospital stay (12.73 ± 11.38; 95% CI: 5.08-20.38) in the above group. Multivariate analysis revealed that nonsurvivors are highest (81%) in (DM + HTN + CAD + CKD) category with an odds ratio (95% CI) of 44.03 (8.64-224.27). Age, gender, and comorbidities adjusted odds ratio decreased to 20.25 (3.77-108.77). Median survival of 7 days was observed in the (DM + HTN + CAD + CKD) category. In summary, the presence of underlying comorbidities has contributed to a higher mortality rate, greater risk of severe disease, and extended hospitalization periods, hence, resulting in overall poorer clinical outcomes in hospitalized COVID-19 patients.

Exosomal PTEN as a Predictive Marker of Aggressive Gliomas.

Background: Liquid biopsies have emerged as convenient alternative diagnostic methods to invasive biopsies, by evaluating disease-specific biomarkers and monitoring the disease risk noninvasively. Phosphatase and tensin homolog deleted in chromosome 10 (PTEN) is a potent tumor suppressor, and its deletion/mutations are common in gliomas. Objective: Evaluate the feasibility of non-invasive detection of PTEN and its downstream genes in serum exosomes of glioma patients. Materials and methods: PTEN, Yes-associated-protein 1 (YAP1), and lysyl oxidase (LOX) transcript expression were monitored through polymerase chain reaction (PCR) in serum exosomes and their paired tumor tissues. The impact of PTEN and its axis genes expression on the overall survival (OS) was monitored. Results: Out of the 106 glioma serum samples evaluated, PTEN was retained/lost in 65.4%/34.6% of the tumor samples while it was retained/lost in 67.1%/32.9% of their paired exosomal fractions. PTEN expression in both tissue and paired exosomal fractions was observed in 48.11% of the samples. Sanger sequencing detected three mutations (Chr10: 89720791(A>G), Chr10:89720749(C>T), and Chr10:89720850(A>G). Both PTEN-responsive downstream genes (YAP1) and LOX axis were upregulated in the PTEN-deficient samples. PTEN loss was associated with poor survival in the glioma patients (hazard ratio (HR) 0.68, confidence interval (CI): 0.35-1.31, P = 0.28). The OS of the exosomal PTEN cohort coincided with the tumor-tissue PTEN devoid group (HR 1.08, CI: 0.49-2.36, P = 0.85). While, old age yielded the worst prognosis; gender, location, and grade were not prognostic of OS in the multivariate analysis. Conclusions: PTEN and its responsive genes YAP1 and LOX can be detected in serum exosomes and can serve as essential tools for the non-invasive evaluation/identification of aggressive gliomas.

Diagnostic Values of Laboratory Biomarkers in Predicting a Severe Course of COVID-19 on Hospital Admission.

Objective: We intend to identify differences in the clinicodemographic and laboratory findings of COVID-19 patients to predict disease severity and outcome on admission. Methods: This single-centred retrospective study retrieved laboratory and clinical data from 350 COVID-19 patients on admission, represented as frequency tables. A multivariate regression model was used to assess the statistically significant association between the explanatory variables and COVID-19 infection outcomes, where adjusted odds ratio (AOR), p value, and 95% CI were used for testing significance. Results: Among the 350 COVID-19 patients studied, there was a significant increase in the WBC count, neutrophils, aggregate index of systemic inflammation (AISI), neutrophil-to-lymphocyte ratio (dNLR), neutrophil-to-lymphocyte and platelet ratio (NLPR), monocyte-to-lymphocyte ratio (MLR), systemic immune-inflammation index (SII), systemic inflammation response index (SIRI), D-dimer, interleukin-6 (IL-6), ferritin, lactate dehydrogenase (LDH), prothrombin time (PT), glucose, urea, urea nitrogen, creatinine, alanine phosphatase (ALP), and aspartate aminotransferase (AST) and a significant decrease in lymphocytes, eosinophils, total protein, albumin, prealbumin serum, and albumin/globulin (A/G) ratio in the severe group when compared with the mild and moderate groups. However, after adjusting their age, gender, and comorbidities, WBC count (adjusted odds ratio (AOR) = 6.888, 95% CI = 1.590-29.839, p = 0.010), neutrophils (AOR = 5.912, 95% CI = 2.131-16.402, p = 0.001), and urea (AOR = 4.843, 95% CI = 1.988-11.755, p = 0.001) were strongly associated with disease severity. Interpretation and Conclusion. On admission, WBC count, neutrophils, and urea, with their cut of values, can identify at-risk COVID-19 patients who could develop severe COVID-19.

Naltrexone Reverses Ethanol Preference and Protein Kinase C Activation in Drosophila melanogaster.

Alcohol use disorder (AUD) is a major health, social and economic problem for which there are few effective treatments. The opiate antagonist naltrexone is currently prescribed clinically with mixed success. We have used naltrexone in an established behavioral assay (CAFE) in Drosophila melanogaster that measures the flies' preference for ethanol-containing food. We have confirmed that Drosophila exposed to ethanol develop a preference toward this drug and we demonstrate that naltrexone, in a dose dependant manner, reverses the ethanol-induced ethanol preference. This effect is not permanent, as preference for alcohol returns after discontinuing naltrexone. Additionally, naltrexone reduced the alcohol-induced increase in protein kinase C activity. These findings are of interest because they confirm that Drosophila is a useful model for studying human responses to addictive drugs. Additionally because of the lack of a closely conserved opiate system in insects, our results could either indicate that a functionally related system does exist in insects or that in insects, and potentially also in mammals, naltrexone binds to alternative sites. Identifying such sites could lead to improved treatment strategies for AUD.

AMPK inhibits MTDH expression via GSK3ß and SIRT1 activation: potential role in triple negative breast cancer cell proliferation.

Recent studies have highlighted the involvement of metadherin (MTDH), an oncogenic protein, in promoting cancer progression, metastasis and chemoresistance in many cancers including mammary carcinomas. However, the molecular regulation of MTDH is still not completely understood. In this study we document that AMP activated protein kinase (AMPK) activation-induced anti-proliferative effects are, in part, mediated by inhibiting MTDH expression in MDA-MB-231 and BT-549 triple negative breast cancer (TNBC) cells. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, caused growth arrest, inhibition of migration and invasion of TNBC cells. Intriguingly, AICAR or metformin treatment resulted in significant downregulation of MTDH expression via inhibiting c-Myc expression. In contrast, treatment of cells with compound C, an inhibitor of AMPK, increased both c-Myc and MTDH expressions in TNBC cells. Also, AMPK activation caused increased glycogen synthase kinase 3ß (GSK3ß) activity by inhibiting the inactive phosphorylation at Ser9, on the one hand, and activation of sirtuin1 (SIRT1) by inhibiting Ser47 phosphorylation, as evidenced by deacetylation of p53, on the other hand. Moreover, AMPK-induced GSK3ß and SIRT1 activities were found to be responsible for inhibiting c-Myc-mediated upregulation of MTDH, as LiCl (an inhibitor of GSK3ß) and EX-527 (an inhibitor of SIRT1) reversed AICAR-mediated downregulation of c-Myc and MTDH expressions. Similar results were observed with siSIRT1 treatment. Furthermore, AICAR and EX-527 treatments caused increased cell death under MTDH-depleted conditions. Finally, we uncovered a novel regulation of MTDH expression and showed that AMPK activation by inducing GSK3ß and SIRT1 downregulates MTDH expression via inhibiting c-Myc in TNBC cells.