Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

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

Toward characterizing extracellular vesicles at a single-particle level

Abstract Extracellular vesicles (EVs) are cell-derived membrane-bound vesicles that serve a means of cell-cell communication. Studying EVs at a single-particle level is important because EVs are inherently heterogeneous. Novel micro- and nanotechnological tools have open opportunities for realizing single-EV measurements exploiting their biochemical, electrical, mechanical, and/or optical properties. This review summarizes the recent development of technologies toward sorting and analyzing single EVs. Sorting EVs into a more homogeneous subset relaxes the sensitivity and throughput required on the EV detection, and hence related techniques are also included in this review. These exciting technologies are on the rise and will expand our understanding of EVs and their applications in the near future

Stability of enzyme immobilized on the nanofluidic channel surface

The lifetime of an enzyme is critical to prevent system failure and optimize maintenance schedules in biological and analytical chemistry. The lifetime metrics of an enzyme can be evaluated from enzyme activity in terms of catalytic cycles per enzyme at various storage times. Trypsin, which is a gold-standard enzyme in proteomics, has been known to decrease activity due to self-digestion. To improve the activity of trypsin, enzyme reactors have developed by immobilizing in micro and nanospace. However, an evaluation method for the catalytic cycle has not been established due to major issues such as nonuniform space, unstable liquid transport, and self-digestion during immobilization in conventional work. To solve these issues, we have previously developed an ultra-fast enzyme reactor with a well-defined nanofabrication method, stable liquid transport, and partial enzyme modification. Here, we aimed to investigate catalytic cycles in a nanochannel. To extend enzyme lifetime efficiently, we have evaluated the optimal immobilization process and catalytic cycles of trypsin. As a result, immobilized enzyme densities by the trypsinogen immobilization process were increased at all concentrations compared to the trypsin immobilization process. To evaluate the lifetime of trypsin, the immobilized enzyme densities and activities were almost the same before and after 72 h of enzyme storage, and the calculated catalytic cycles were 1740. These results indicated that self-digestion of the immobilized enzyme was highly suppressed. Consequently, the reaction efficiency has been evaluated depending on the catalytic cycles from the substrate for the first time, while preventing self-digestion by trypsin

Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules

A High-throughput automated microfluidic platform for calcium imaging of taste sensing

[[abstract]]The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca2+ concentration. However, glucose evoked a rapid elevation of intracellular Ca2+ followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening

Effects of music listening on stress, anxiety and sleep quality for sleep disturbed pregnant women

Prenatal sleep disturbance has been associated with undesirable birthing outcomes. To determine the effectiveness of listening to music at home in improving sleep quality, 121 Taiwanese pregnant women with poor sleep quality (Pittsburgh Sleep Quality Index [PSQI] score \u3e 5) were systematically assigned, with a random start to music listening (n = 61) or control (n = 60) group. Participants in the music listening group self-regulated listening to music in addition to receiving general prenatal care similar to that in the control group for 2 weeks. The PSQI and State-Anxiety Inventory were used to assess outcomes. ANCOVA analyses were used with the pretest scores as covariates and showed significant improvement in sleep quality, stress, and anxiety in the music listening group compared with the control group. The most frequently used music genre by participants in the experimental group was lullabies, followed by classical music and crystal baby music. This study supported the theory that 2-week music listening interventions may reduce stress, anxiety, and yield better sleep quality for sleep-disturbed pregnant women. The analysis of participants’ journals also implied that the expectant mothers’ choices of musical genres may correlate more with perceived prenatal benefits or the desire to interact with their unborn child

Fused silica microchannel fabrication with smooth surface and high etching selectivity

Channel fabrication technology has become increasingly important for microfluidic and nanofluidic devices. In particular, glass channels have high chemical and physical stability, high optical transparency, and ease of surface modification, so that there is increasing interest in glass microfluidic devices for chemical experiments in microfluidics and nanofluidics. For the fabrication of glass channels, especially those with a high aspect ratio (depth/width), lithography using a metal resist and dry etching have mainly been used. However, there are still issues involving the surface roughness of the etched channel and the low etching selectivity. In this study, a microchannel fabrication method with high etching selectivity that produces a smooth etched surface was developed. First, interference during dry etching by remaining Cr particles after the photolithography and Cr etching processes was assumed as the cause of the rough etched surface. Three different dry etching processes were introduced to verify this. In process 1 without removal of the Cr particles, the etched surface was not flat and had a 1 μm scale roughness. In process 2 where a cleaning process was included and high power etching was conducted, a smooth surface with a 1 nm scale roughness and a faster etching rate of 0.3 μm min−1 were obtained. For this high-power etching condition, the etching selectivity (fused silica/Cr) was relatively low at approximately 39-43. In process 3 with a cleaning process and low-power etching, although the etching rate was relatively low at 0.1 μm min−1, a smooth surface with 1 nm scale roughness (10 nm scale roughness deeper than 40 μm in the depth region) and a much higher etching selectivity of approximately 79-84 were obtained. The dry etching method presented in this study represents a significant contribution to microfluidics/nanofluidics for microchannel/nanochannel fabrication