The microRNA (miRNA) cargo contained in plasma extracellular vesicles (EVs) offers a relatively little explored source of biomarkers for brain diseases that can be obtained noninvasively. Methods to isolate EVs from plasma, however, are still being developed. For EV isolation, it is important to ensure the removal of vesicle-free miRNAs, which account for approximately two-thirds of plasma miRNAs. Membrane particle precipitation-based EV isolation is an appealing method because of the simple protocol and high yield. Here, we evaluated the performance of a precipitation-based method to obtain enriched EV-specific miRNAs from a small volume of rat plasma. We performed size-exclusion chromatography (SEC) on precipitation-isolated EV pellets and whole plasma. The SEC fractions were analysed using Nanoparticle Tracking Analysis (NTA), protein and miRNA concentration assays, and droplet digital polymerase chain reaction for four miRNAs (miR-142-3p, miR-124-3p, miR-23a, miR-122). Precipitation-isolated EVs and selected SEC fractions from the plasma were also analysed with transmission electron microscopy (TEM). Precipitation-based EV isolation co-precipitated 9% to 15% of plasma proteins and 21% to 99% of vesicle-free miRNAs, depending on the individual miRNAs. In addition, the amount of miR-142-3p, found mainly in EV fractions, was decreased in the EV fractions, indicating that part of it was lost during precipitation-based isolation. Western blot and TEM revealed both protein and lipoprotein contamination in the precipitation-isolated EV-pellets. Our findings indicate that a precipitation-based method is not sufficient for purifying plasma EV-contained miRNA cargo. The particle number measured by NTA is high, but this is mostly due to the contaminating lipoproteins. Although a part of the vesicle-free miRNA is removed, vesicle-free miRNA still dominates in plasma EV pellets isolated by the precipitation-based method.

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Introduction:In the conventional system of medical education, basic subjects are taught in the 1st year with least interdisciplinary interaction. The objective of this study was to explore the students’ perception about content, need and application of basic science subjects during the clinical years of their medical education.Materials and Methods:A total of 300 questionnaires were distributed among students randomly after taking their written consent for participation in the study. About 265 completely filled questionnaires were received back and the response was analyzed.Results:Students identified anatomy as the subject with overloaded syllabus (75.4%) and also with maximum clinical application with 50.1% of them considering it the most important basic subject. Students were satisfied with the practical integration of subjects to impart clinical skills, but considered problem based learning a better method of teaching. According to 37%, 43.8% and 33.2% of respondents respectively; anatomy, biochemistry and physiology curriculum should only cover the general concepts to give the working knowledge of the subject. Approximately, 65% of the respondents were able to recall the knowledge of anatomy and physiology while biochemistry was retained by 40%.Conclusions:Overall, the attitudes of students toward basic science subjects were positive. The learning experience for them can be improved significantly by better clinical integration of the subjects.

The World Health Organization estimates that globally 2.4 million people are diagnosed with epilepsy each year. In nearly 30% of these cases, epilepsy cannot be properly controlled by antiepileptic drugs. More importantly, treatments to prevent or modify epileptogenesis do not exist. Therefore, novel therapies are urgently needed. In this respect, it is important to identify which patients will develop epilepsy and which individually tailored treatment is needed. However, currently, we have no tools to identify the patients at risk, and diagnosis of epileptogenesis remains as a major unmet medical need, which relates to lack of diagnostic biomarkers for epileptogenesis. As the epileptogenic process in humans is typically slow, the use of animal models is justified to speed up biomarker discovery. We aim to summarize recommendations for molecular biomarker research and propose a standardized procedure for biomarker discovery in rat models of epileptogenesis. The potential of many phylogenetically conserved circulating noncoding small RNAs, including microRNAs (miRNAs), as biomarkers has been explored in various brain diseases, including epilepsy. Recent studies show the feasibility of detecting miRNAs in blood in both experimental models and human epilepsy. However, the analysis of circulating miRNAs in rodent models is challenging, which relates both to the lack of standardized sampling protocols and to analysis of miRNAs. We will discuss the issues critical for preclinical plasma biomarker discovery, such as documentation, blood and brain tissue sampling and collection, plasma separation and storage, RNA extraction, quality control, and RNA detection. We propose a protocol for standardization of procedures for discovery of circulating miRNA biomarkers in rat models of epileptogenesis. Ultimately, we hope that the preclinical standardization will facilitate clinical biomarker discovery for epileptogenesis in man.

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