Sleep is required to consolidate odor memory and remodel olfactory synapses

Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here, we show that, although the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear if, in any system, sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures

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

Schizophrenia-associated chromosome 11q21 translocation: Identification of flanking markers and development of chromosome 11q fragment hybrids as cloning and mapping resources

Genetic linkage, molecular analysis, and in situ hybridization have identified TYR and D11S388 as markers flanking the chromosome 11 breakpoint in a large pedigree where a balanced translocation, t(1;11)(q43;q21), segregates with schizophrenia and related affective disorders. Somatic cell hybrids, separating the two translocation chromosomes from each other and from the normal homologues, have been produced with the aid of immunomagnetic sorting for chromosome 1– and chromosome 11–encoded cell-surface antigens. The genes for two of these antigens map on either side of the 11q breakpoint. Immunomagnetic bead sorting was also used to isolate two stable X-irradiation hybrids for each cell-surface antigen. Each hybrid carries only chromosome 11 fragments. Translocation and X-irradiation hybrids were analyzed, mainly by PCR, for the presence of 19 chromosome 11 and 4 chromosome 1 markers. Ten newly designed primers are reported. The X-irradiation hybrids were also studied cytogenetically, for human DNA content, by in situ Cot1 DNA hybridization and by painting the Alu-PCR products from these four lines back onto normal human metaphases. The generation of the translocation hybrids and of the chromosome 11q fragment hybrids is a necessary preliminary to determining whether a schizophrenia-predisposition gene SCZD2 is encoded at this site

TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia and dysregulation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease based on transgenic mouse studies. To determine a role of microRNAs on the pathogenesis of the aggressive form of CLL we studied regulation of TCL1 expression in CLL by microRNAs. We identified miR-3676 as a regulator of TCL1 expression. We demonstrated that miR-3676 targets three consecutive 28-bp repeats within 3'UTR of TCL1 and showed that miR-3676 is a powerful inhibitor of TCL1. We further showed that miR-3676 expression is significantly down-regulated in four groups of CLL carrying the 11q deletions, 13q deletions, 17p deletions, or a normal karyotype compared with normal CD19(+) cord blood and peripheral blood B cells. In addition, the sequencing of 539 CLL samples revealed five germ-line mutations in six samples (1%) in miR-3676. Two of these mutations were loss-of-function mutations. Because miR-3676 is located at 17p13, only 500-kb centromeric of tumor protein p53 (Tp53), and is codeleted with Tp53, we propose that loss of miR-3676 causes high levels of TCL1 expression contributing to CLL progression

Haploinsufficiency of the Sec7 Guanine Nucleotide Exchange Factor <em>Gea1</em> Impairs Septation in Fission Yeast

<div><p>Membrane trafficking is essential to eukaryotic life and is controlled by a complex network of proteins that regulate movement of proteins and lipids between organelles. The GBF1/GEA family of Guanine nucleotide Exchange Factors (GEFs) regulates trafficking between the endoplasmic reticulum and Golgi by catalyzing the exchange of GDP for GTP on ADP Ribosylation Factors (Arfs). Activated Arfs recruit coat protein complex 1 (COP-I) to form vesicles that ferry cargo between these organelles. To further explore the function of the GBF1/GEA family, we have characterized a fission yeast mutant lacking one copy of the essential gene <em>gea1</em> (<em>gea1</em>+/−), the <em>Schizosaccharomyces pombe</em> ortholog of <em>GBF1</em>. The haploinsufficient <em>gea1</em>+/− strain was shown to be sensitive to the GBF1 inhibitor brefeldin A (BFA) and was rescued from BFA sensitivity by gea1p overexpression. No overt defects in localization of arf1p or arf6p were observed in <em>gea1+/−</em> cells, but the fission yeast homolog of the COP-I cargo sac1 was mislocalized, consistent with impaired COP-I trafficking. Although Golgi morphology appeared normal, a slight increase in vacuolar size was observed in the <em>gea1</em>+/− mutant strain. Importantly, <em>gea1</em>+/− cells exhibited dramatic cytokinesis-related defects, including disorganized contractile rings, an increased septation index, and alterations in septum morphology. Septation defects appear to result from altered secretion of enzymes required for septum dynamics, as decreased secretion of eng1p, a β-glucanase required for septum breakdown, was observed in <em>gea1</em>+/− cells, and overexpression of eng1p suppressed the increased septation phenotype. These observations implicate <em>gea1</em> in regulation of septum breakdown and establish <em>S. pombe</em> as a model system to explore GBF1/GEA function in cytokinesis.</p> </div

Primers used in this study.

<p>Primers used in this study.</p

COP-I-dependent transport is impaired in <i>gea1+/−</i> cells.

<p>A. Wild-type and <i>gea1+/−</i> cells were transformed with pDUAL-YFH1c-<i>arf1</i> or pDUAL-YFH1c-<i>arf6</i> and imaged by fluorescence microscopy. Scale bar, 10 µM. B. Wild-type and <i>gea1+/−</i> cells were transformed with pDUAL-YFH1c-<i>sac11</i> (SPBC19F5.03) or pDUAL-YFH1c-<i>sac12</i> (SPAC3C7.01c) and imaged by fluorescence microscopy. Scale bar, 10 µM. C. Localization of sac11-YFP in wild-type (n = 207) and <i>gea1+/−</i> cells (n = 110) was scored as ER (surrounding the cell cortex and nuclear envelope), Golgi (punctate in the cytoplasm), or mixed. Sac11-YFP was predominately found in the ER in wild-type cells and in the Golgi and mixed in <i>gea1+/−</i> cells. D. Localization of sac12-YFP in wild-type (n = 204) and <i>gea1+/−</i> cells (n = 147) was scored as ER, Golgi, or mixed. Sac11-YFP was predominately found in the Golgi in both wild-type and <i>gea1+/−</i> cells. Error bars represent the mean ± SD from 3 independent experiments.</p

Organellar morphology in <i>gea1+/−</i> cells.

<p>A. Wild-type and <i>gea1+/−</i> cells were stained with 5 µM BODIPY FL C₅-ceramide to label the Golgi or with 32 µM FM4-64 to label the vacuole. Staining was visualized by fluorescence microscopy. Scale bar, 10 µM. B. The pixel area associated with the largest vacuole was measured using Image J for individual wild-type (n = 262) and <i>gea1+/−</i> (n = 225) cells. The percentage of cells with vacuolar sizes of the indicated ranges was plotted using SigmaPlot. C. Wild-type (A) and <i>gea1</i>+/− cells (B) were subjected to transmission electron microscopy to visualize membranous structures. Representative images show flat ribbon-like structures consistent with Golgi membranes (insets) that appear similar in wild-type and <i>gea1+/−</i> cells. N, nucleus. Scale bar, 500 nm. D. Cells were labeled with FM4-64, followed by incubation in H₂O for 90 min to induce vacuolar fusion. Scale bar, 10 µM.</p

<i>Gea1+/−</i> cells exhibit alterations in septum number and morphology.

<p>A. Wild-type and <i>gea1</i>+/− cells were stained with calcofluor white to visualize septa and imaged by fluorescence microscopy. Scale bar, 10 µM. B. Quantification of A. Cells were scored as having abnormal septa if multiple septa were present, or if the septum was mislocalized, abnormally thick, or forked. Error bars represent mean ± SD from 3 independent experiments. C. Wild-type and <i>gea1</i>+/− cells were subjected to transmission electron microscopy to visualize septum defects. Representative images of multi-septated cells and septa with morphological abnormalities are shown. Scale bar, 1 µm.</p