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

Frequency and Specificity of Red Blood Cell Alloimmunization in Chilean Transfused Patients

Background: Alloimmunization is an adverse effect of blood transfusions. In Chile, alloimmunization frequency is not established, and for this reason the aim of this study was to investigate the prevalence and specificity of red blood cell (RBC) alloantibodies in Chilean transfused subjects. Methods: Records from 4,716 multi-transfused patients were analyzed. In these patients, antibody screening was carried out prior to cross-matching with a commercially available two-cell panel by the microcolum gel test, and samples with a positive screen were analyzed for the specificity of the alloantibody with a 16-cell identification panel. Results: The incidence of RBC alloimmunization in transfused patients was 1.02% (48/4,716) with a higher prevalence in women (40/48). We detected 52 antibodies, the most frequent specificities identified were anti-E (30.8%), anti-K (26.9%), anti-D (7.7%), and anti-Fy(a) (5.8%). The highest incidence of alloantibodies was observed in cancer and gastroenterology patients. Conclusion: The data demonstrated a low alloimmunization frequency in Chilean transfused patients, principally associated with antibodies anti-E, anti-K, anti-D, and anti-Fy(a)

Glutamate counteracts Dopamine/PKA signaling via dephosphorylation of DARPP-32 Ser-97 and alteration of its cytonuclear distribution

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels

Hyccin expression levels in organotypic DRG cultures.

<p>Panel A: Cells were maintained in culture for the times indicated and analysis of MPZ mRNA was utilized as a reference for myelination in the culture. Results are expressed as mRNA fold increase relative to calibrator (10 days) normalized to housekeeping genes (b-actin and GAPDH) For each sample data are represented as means ±SEM (n = 3) and .p values were <0.05 compared to control. Panel B: Total cellular lysates were isolated at the times indicated and subjected to immunoblot analysis for the myelin markers MPZ and MBP. An antibody against GAPDH was used as an internal control. Panel C: At 30 days cell cultures were fixed, stained with 1% Sudan Black in 70% ethanol and analyzed by optic microscopy. Bright-field micrographs confirm the presence of myelin sheaths with Ranvier nodes (white arrows) surrounding th axons.</p

Analysis of β-galactosidase activity in the CNS from P2 (A,B,C), P10 (D,E,F), P30 (G,H,I) hyccin Het mice (n = 3).

<p>In the CNS, Hyccin is expressed in olfactory nuclei (A,D,G), in distinct layers of the neocortex (arrows in B,C, E,H) and in the hippocampal region with stronger expression in CA1 and CA2 fields as compared to CA3 (F,I arrows); dentate gyrus is negative. Other sites of expression include the prefrontal cortex, the piriform cortex (arrowheads B and D) and the ventromedial hypothalamic nuclei (I, arrowhead). Scale bars are 1000 micron (A,–F) and 2000 micron (G–I).</p

RNA in situ hybridization on brain sections from P30 WT mice reveals an expression pattern in agreement with principal neuronal expression.

<p>Coronal brain sections were hybridized with a Hyccin antisense 700 bp probe. Panel A: High-magnification image of hippocampus shows the strongest labeling of Hyccin in the CA1, 2 region compared to the CA3 region and dentate gyrus (DG). Scale bar is 200 µ. Panel B: High-magnification image of the cerebral cortex revealed a stronger labeling of Hyccin in deep cortical layers (V/VI) compared to superficial layers (II–III). Scale bar is 200 µ.</p

Immunofluorescence analysis of hyccin expression in DRG neurons and Schwann cell cultures.

<p>Panel A: Immunocolocalization of GFP-Hyccin transiently transfected in Hela cells and the anti-hyccin polyclonal antibody. Representative images of Hela cells transfected with GFP-Hyccin and immunostained with anti-hyccin polyclonal antibody 24 hr after transfection. Hyccin immunoreactivity was detected by Alexa546 conjugated secondary antibodies. The arrows in the merge panel show colocalization of the two signals in the cytosol. Final magnification 63×. Scale bars represent 100 µ. Panel B: DRG neurons cultures were immunostained with an anti-hyccin antibody and an anti-phosphorylated neurofilament. Hyccin and Neurofilament immunoreactivities were detected respectively by Alexa488 and Alexa546 conjugated secondary antibodies. The merge panel shows positivity of the two signals in the neurites. Final magnification 40×. Panel C: Schwann cells cultures were immunostained with an anti-hyccin polyclonal antibody and an anti-S100 monoclonal antibody. Hyccin and S100 immunoreactivities were detected respectively by Alexa488 and Alexa546 conjugated secondary antibodies. Final magnification 100×.</p

Real-time PCR analysis of Hyccin transcripts in total brain lysates from P2 (n = 12), P10 (n = 12), P30 (n = 6) and P60 (n = 6) WT mice.

<p>Results are expressed as mRNA fold increase relative to calibrator (p2) normalized to housekeeping genes (b-actin and GAPDH). Quantitative data were reported as median values with first and third quartiles (1st-3rd q) and minimum and maximum value (Min-max). * = p<0.01.</p

Generation of mice expressing lacZ under control of the Hyccin gene regulatory elements.

<p>Panel A: Schematic representation of the <i>Fam126a</i> locus and engineering of a <i>Fam126a</i> -LacZ allele. A segment containing exons 2 and 3 were replaced with a cassette containing a modified β-galactosidase coding sequence (LacZ) and a drug resistance minigene comprised of the phosphoglycerate kinase promoter (PGKp), a bacterial promoter (EM7p) and neomycin phosphotransferase (Neor), flanked by loxP sequences. The numbers below the line representing the genomic sequence indicate base pairs. Panel B: A representative PCR analysis of tail genomic DNA from wild-type (WT), heterozygous (Het) homozygous LacZ knockin mice and documenting <i>Fam126a</i> gene deletion.</p