Investigating the Potential and Pitfalls of EV-Encapsulated MicroRNAs as Circulating Biomarkers of Breast Cancer

Extracellular vesicles (EVs) shuttle microRNA (miRNA) throughout the circulation and are believed to represent a fingerprint of the releasing cell. We isolated and characterized serum EVs of breast tumour-bearing animals, breast cancer (BC) patients, and healthy controls. EVs were characterized using transmission electron microscopy (TEM), protein quantification, western blotting, and nanoparticle tracking analysis (NTA). Absolute quantitative (AQ)-PCR was employed to analyse EV-miR-451a expression. Isolated EVs had the appropriate morphology and size. Patient sera contained significantly more EVs than did healthy controls. In tumour-bearing animals, a correlation between serum EV number and tumour burden was observed. There was no significant relationship between EV protein yield and EV quantity determined by NTA, highlighting the requirement for direct quantification. Using AQ-PCR to relate miRNA copy number to EV yield, a significant increase in miRNA-451a copies/EV was detected in BC patient sera, suggesting potential as a novel biomarker of breast cancer

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.Highlights: Harmonized QA/QC procedures for SARS-CoV-2 wastewater surveillance are lacking; Wastewater analysis protocols are not optimized for trace analysis of viruses; False-positive and -negative errors have consequences for public health responses; Inter-laboratory studies utilizing standardized reference materials and protocols are needed.info:eu-repo/semantics/publishedVersio

Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C

BACKGROUND: Punica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C. METHODS: PRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. RESULTS: The screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus. CONCLUSION: This is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations

Antimicrobial activities of pomegranate rind extracts: enhancement by addition of metal salts and vitamin C

Abstract Background Punica granatum L. or pomegranates, have been reported to have antimicrobial activity against a range of Gram positive and negative bacteria. Pomegranate formulations containing ferrous salts have enhanced although short-term, antibacteriophage activities which are rapidly diminished owing to instability of the ferrous combination. The aim of this study was to determine the antimicrobial activities of combinations of pomegranate rind extracts (PRE) with a range of metals salts with the added stabiliser vitamin C. Methods PRE solutions, prepared by blending rind sections with distilled water prior to sterilisation by autoclaving or filtration, were screened with a disc diffusion assay using penicillin G as a control. Suspension assays were used to determine the antimicrobial activities of PRE alone and in combination with salts of the following metals; Fe (II), Cu (II), Mn (II) or Zn (II), and vitamin C, against a panel of microbes following exposure for 30 mins. The test organisms included Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis. Results The screening assay demonstrated that PRE exhibited activity against the Gram positive organisms at 24 h with no observable effect on any of the Gram negative bacteria. However, after 12 h, zones of inhibition were only observed for Ps. aeruginosa. In contrast, using the suspension assay, addition of Cu (II) salts to PRE solutions extended the activities resulting in no detectable growth being observed for the PRE/Cu (II) combination against E. coli, Ps. aeruginosa and P. mirabilis. Minimal antimicrobial activity was observed following incubation with Fe (II), Mn (II) or Zn (II) salts alone or in combination with PRE against any of the organisms in the test panel. The addition of vitamin C markedly enhanced the activities of both PRE/Fe (II) and PRE/Cu (II) combinations against S. aureus. Conclusion This is the first report demonstrating the enhanced efficacy of PRE/metal salt combinations in the presence of the stabilising agent vitamin C, to which all isolates were sensitive with the exception of B. subtilis. This study has validated the exploration of PRE along with additives such as metal salts and vitamin C as novel antimicrobial combinations.</p