The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
When an otherwise harmful insult to the brain is preceded by a brief, noninjurious stimulus, the brain becomes tolerant, and the resulting damage is reduced. Epileptic tolerance develops when brief seizures precede an episode of prolonged seizures (status epilepticus). MicroRNAs (miRNAs) are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. We investigated how prior seizure preconditioning affects the miRNA response to status epilepticus evoked by intra-amygdalar kainic acid in mice. The miRNA was extracted from the ipsilateral CA3 subfield 24 hours after focal-onset status epilepticus in animals that had previously received either seizure preconditioning (tolerance) or no preconditioning (injury), and mature miRNA levels were measured using TaqMan low-density arrays. Expression of 21 miRNAs was increased, relative to control, after status epilepticus alone, and expression of 12 miRNAs was decreased. Increased miR-132 levels were matched with increased binding to Argonaute-2, a constituent of the RNA-induced silencing complex. In tolerant animals, expression responses of >40% of the injury-group-detected miRNAs differed, being either unchanged relative to control or down-regulated, and this included miR-132. In vivo microinjection of locked nucleic acid-modified oligonucleotides (antagomirs) against miR-132 depleted hippocampal miR-132 levels and reduced seizure-induced neuronal death. Thus, our data strongly suggest that miRNAs are important regulators of seizure-induced neuronal death.
MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.MicroRNAs (miRNAs) are a large class of noncoding RNAs involved in posttranscriptional regulation through RNA interference. A number of recent studies have identified virally encoded miRNAs using either biochemical cloning strategies, bioinformatics, or a combination of the two approaches (1, 2, 4, 7-9). In this study an alternative bioinformatics approach based on comparative conservation between predicted stemloop sequences of human cytomegalovirus (HCMV) and chimpanzee cytomegalovirus (CCMV) was used to predict candidate miRNAs. Expression of predicted miRNAs during HCMV infection was then assessed by Northern blot analysis. Our bioinformatics approach utilized a computer algorithm called Stem-loop Finder (SLF; Combimatrix) to predict potential RNA transcripts from the HCMV genome that could form stem-loop secondary structures. The algorithm uses free energy calculations to determine the theoretical stability of the base pairing within the stem region, including pairing between G · U bases, while maintaining a maximum and minimum length between the complementary base pairing to determine loop size. A scoring matrix then weights beneficial or detrimental folding structures and attributes a cumulative score to each potential stem-loop sequence. Analysis of the HCMV genome using SLF identified 406 potential stem-loop sequences. Previously identified miRNAs are often extensively conserved between many different species (6). Consequently, we hypothesized that functionally important miRNAs expressed by HCMV would be conserved between HCMV and closely related viruses, such as CCMV. The sequence of each of the 406 candidate SLF-derived HCMV stem-loop transcripts was compared with the CCMV genome for potential homology. A minimum score of 60% homology with CCMV was used to select stem-loop sequences for further analysis. Our preliminary studies determined that this level of homology was sufficiently stringent to identify significant sequence conservation without exclusion of any potential miRNAs. Of the 406 sequences analyzed, 110 potential stem-loop sequences scored higher than 60% homology.The 110 HCMV stem-loop sequences selected using the cri...
MicroRNAs are small RNAs that function as regulators of posttranscriptional gene expression. MicroRNAs are encoded by genes, and processed to form ribonucleoprotein complexes that bind to messenger RNA (mRNA) targets to repress translation or degrade mRNA transcripts. The microRNAs are particularly abundant in the brain where they serve as effectors of neuronal development and maintenance of the neuronal phenotype. They are also expressed in dendrites where they regulate spine structure and function as effectors in synaptic plasticity. MicroRNAs have been evaluated for their roles in brain ischemia, traumatic brain injury, and spinal cord injury, and in functional recovery after ischemia. They also serve as mediators in the brain’s response to ischemic preconditioning that leads to endogenous neuroprotection. In addition, microRNAs are implicated in neurodegenerative disorders, including Alzheimer’s, Huntington, Parkinson, and Prion disease. The discovery of microRNAs has expanded the potential for human diseases to arise from genetic mutations in microRNA genes or sequences within their target mRNAs. This review discusses microRNA discovery, biogenesis, mechanisms of gene regulation, their expression and function in the brain, and their roles in brain ischemia and injury, neuroprotection, and neurodegeneration.
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