Gut Microbiota Patterns in Patients with Non-Alcoholic Fatty Liver Disease: A Comprehensive Assessment Using Three Analysis Methods.

Non-alcoholic fatty liver disease (NAFLD) is considered the most common chronic liver disease worldwide, affecting nearly 25% of the global adult population. Increasing evidence suggests that functional and compositional changes in the gut microbiota may contribute to the development and promote the progression of NAFLD. 16S rRNA gene next-generation sequencing is widely used to determine specific features of the NAFLD microbiome, but a complex system such as the gut microbiota requires a comprehensive approach. We used three different approaches: MALDI-TOF-MS of bacterial cultures, qPCR, and 16S NGS sequencing, as well as a wide variety of statistical methods to assess the differences in gut microbiota composition between NAFLD patients without significant fibrosis and the control group. The listed methods showed enrichment in Collinsella sp. and Oscillospiraceae for the control samples and enrichment in Lachnospiraceae (and in particular Dorea sp.) and Veillonellaceae in NAFLD. The families, Bifidobacteriaceae, Lactobacillaceae, and Enterococcaceae (particularly Enterococcus faecium and Enterococcus faecalis), were also found to be important taxa for NAFLD microbiome evaluation. Considering individual method observations, an increase in Candida krusei and a decrease in Bacteroides uniformis for NAFLD patients were detected using MALDI-TOF-MS. An increase in Gracilibacteraceae, Chitinophagaceae, Pirellulaceae, Erysipelatoclostridiaceae, Muribaculaceae, and Comamonadaceae, and a decrease in Acidaminococcaceae in NAFLD were observed with 16S NGS, and enrichment in Fusobacterium nucleatum was shown using qPCR analysis. These findings confirm that NAFLD is associated with changes in gut microbiota composition. Further investigations are required to determine the cause-and-effect relationships and the impact of microbiota-derived compounds on the development and progression of NAFLD.

Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes.

Extracellular circulating microRNAs (miRNAs) are currently a focus of interest as non-invasive biomarkers of cardiovascular pathologies, including coronary artery disease (CAD) and acute coronary syndromes (ACS): myocardial infarction with and without ST-segment elevation (STEMI and NSTEMI) and unstable angina (UA). However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological variances in different studies. In this work, we fulfilled the basic pre-analytical requirements provided for circulating miRNA studies for application to stable CAD and ACS research. We used quantitative PCR to determine the relative plasma levels of eight circulating miRNAs that are potentially associated with atherosclerosis. In a cohort of 136 adult clinic CAD patients and outpatient controls, we found that the plasma levels of miR-21-5p and miR-146a-5p were significantly elevated in ACS patients, and the level of miR-17-5p was decreased in ACS and stable CAD patients compared to both healthy controls and hypertensive patients without CAD. Within the ACS patient group, no differences were found in the plasma levels of these miRNAs between patients with positive and negative troponin, nor were any differences found between STEMI and NSTEMI. Our results indicate that increased plasma levels of miR-146a-5p and miR-21-5p can be considered general ACS circulating biomarkers and that lowered miR-17-5p can be considered a general biomarker of CAD.

Elevated Plasma Levels of Circulating Extracellular miR-320a-3p in Patients with Paroxysmal Atrial Fibrillation.

The potential of extracellular circulating microRNAs (miRNAs) as non-invasive biomarkers of atrial fibrillation (AF) has been confirmed by a number of recent studies. However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological differences. In this work, we attempted to fulfill the basic pre-analytical requirements provided for circulating miRNA studies for application to paroxysmal atrial fibrillation (PAF) research. We used quantitative PCR (qPCR) to determine the relative plasma levels of circulating miRNAs expressed in the heart or associated with atrial remodeling or fibrillation with reported altered plasma/serum levels in AF: miR-146a-5p, miR-150-5p, miR-19a-3p, miR-21-5p, miR-29b-3p, miR-320a-3p, miR-328-3p, miR-375-3p, and miR-409-3p. First, in a cohort of 90 adult outpatient clinic patients, we found that the plasma level of miR-320a-3p was elevated in PAF patients compared to healthy controls and hypertensive patients without AF. We further analyzed the impact of medication therapies on miRNA relative levels and found elevated miR-320a-3p levels in patients receiving angiotensin-converting-enzyme inhibitors (ACEI) therapy. Additionally, we found that miR-320a-3p, miR-21-5p, and miR-146a-5p plasma levels positively correlated with the CHA2DS2-Vasc score and were elevated in subjects with CHA2DS2-Vasc ≥ 2. Our results indicate that, amongst the analyzed miRNAs, miR-320a-3p may be considered as a potential PAF circulating plasma biomarker, leading to speculation as to whether this miRNA is a marker of platelet state change due to ACEI therapy.