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Federica Cavalera, Mario Zanoni, Valeria Merico, Thi Thu Hien Bui, Martina Belli, Lorenzo Fassina, Silvia Garagna, Maurizio Zuccotti
Infertility clinics would benefit from the ability to select developmentally competent vs. incompetent oocytes using non-invasive procedures, thus improving the overall pregnancy outcome. We recently developed a classification method based on microscopic live observations of mouse oocytes during their in vitro maturation from the germinal vesicle (GV) to the metaphase II stage, followed by the analysis of the cytoplasmic movements occurring during this time-lapse period. Here, we present detailed protocols of this procedure...
March 3, 2018: Journal of Visualized Experiments: JoVE
María Andrea Hernández-Castañeda, Smart Mbagwu, Kehinde Adebayo Babatunde, Michael Walch, Luis Filgueira, Pierre-Yves Mantel
Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host...
February 14, 2018: Journal of Visualized Experiments: JoVE
Hoda Fathali, Johan Dunevall, Soodabeh Majdi, Ann-Sofie Cans
Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress...
February 19, 2018: Journal of Visualized Experiments: JoVE
Adriely Goes, Gregor Fuhrmann
Biogenic and biomimetic therapeutics are a relatively new class of systems that are of physiological origin and/or take advantage of natural pathways, or aim at mimicking these to improve selective interaction with target tissue. The number of biogenic and bioengineered avenues for drug therapy and diagnostics has multiplied over the past years for many applications, indicating the high expectations associated with this biological route. Nevertheless, the use of "bio"-related approaches for treating or diagnosing infectious diseases is still rare...
March 19, 2018: ACS Infectious Diseases
Jianying Shen, Jian Zhou, Yong Lin, Zhen Liu, Ping Chen, Xianchun Wang
Plasma membrane (PM) and mitochondrial membrane (MM) proteins of rat hippocampal neurons were identified and comparatively characterized on the basis of a sequential digestion-centered combinative strategy for sample treatment. A total of 478 membrane proteins were identified, of which 240 had PM localization, 170 had MM localization, and 33 had both of the two subcellular localizations. Compared with the PM proteome, the MM proteome not only was smaller, more basic, and more hydrophobic, but also had a narrower protein molecular mass distribution range and a higher proportion of transmembrane proteins...
March 19, 2018: Analytical and Bioanalytical Chemistry
Larisa G Poddubnaya, Willy Hemmingsen, Magdaléna Bruñanská, David I Gibson
This investigation of Calicotyle affinis is the first ultrastructural description of the male reproductive system of a monocotylid monogenean. It has revealed numerous characteristics which are either new or clarify those determined from previous light microscopical observations on the morphology of Calicotyle spp. These include numerous individual testes, each surrounded by its own basal lamina; an ejaculatory bulb delimited by a basal lamina surrounded by two independent internal and external bands of muscles; a bipartite, but not fully divided, muscular chamber (an internal seminal vesicle) in the proximal region of the ejaculatory bulb; well-developed unicellular prostatic glands localised outside the ejaculatory bulb, the cytoplasmic extensions of which pass into the ejaculatory bulb, to form the ducts which open into the lumen of ejaculatory duct; and a male copulatory organ in the form of a coiled penis tube containing throughout its lumen a long, hollow stylet...
March 18, 2018: Parasitology Research
Roman M Lazarenko, Claire E DelBove, Qi Zhang
Release and recycling of synaptic vesicles are essential for neurotransmission and synaptic plasticity. To gain mechanistic understanding of these processes, direct measurements of vesicle release and retrieval is indispensable. Styryl dyes like FM1-43 and FM4-64 have been widely used for this purpose and their loading and unloading are reliable measurements for synaptic vesicle release and retrieval in cultured neurons. This protocol describes in detail the procedure of using styryl dyes to label and measure synaptic vesicle uptake and release in cultured rat hippocampal neurons...
January 20, 2018: Bio-protocol
Xingmin Aaron Zhang, Thomas F J Martin
Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal3 H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al...
January 5, 2018: Bio-protocol
Fengyan Deng, Nancy Magee, Yuxia Zhang
Cell-to-cell communication is a fascinating process that is essential for maintaining tissue and whole-body homeostasis. Extracellular vesicles (EVs) are cell-derived membrane-bound nanoparticles that are a means of communication between cells. Accumulating evidence indicates that EVs can render either beneficial or harmful outcomes, depending on the specific cargos (e.g. proteins, lipids, RNAs) transferred between cells. EVs also have great value as diagnostic and prognostic markers of disease because they are present in a variety of biological fluids and carry bioactive molecules from their cells or tissues of origin...
September 2017: Liver Research
Jin-Peng Wang, Yan-Yan Tang, Chun-Mei Fan, Can Guo, Yan-Hong Zhou, Zheng Li, Xiao-Ling Li, Yong Li, Gui-Yuan Li, Wei Xiong, Zhao-Yang Zeng, Fang Xiong
An increasing number of studies has confirmed that many cells can secrete vesicles or exosomes in eukaryotes, which contain important nucleic acids, proteins and lipids and play important roles in cell communication and tumor metastasis. This paper summarizes the comprehensive function of exosomal non-coding RNAs. Although some studies have shown that exosomes mediate tumor signal transduction, the functional mechanism of the tumor metastasis remains to be elucidated. In this paper, we reviewed the role of exosomal non-coding RNAs in mediating cancer metastasis in the tumor microenvironment to provide new ideas for the study of tumor pathophysiology...
February 23, 2018: Oncotarget
Mandy Berndt-Paetz, Luise Herbst, Annett Weimann, Andreas Gonsior, Jens-Uwe Stolzenburg, Jochen Neuhaus
PURPOSE: Muscarinic acetylcholine receptors (mAChRs) regulate a number of important physiological functions. Alteration of mAChR expression or function has been associated in the etiology of several pathologies including functional bladder disorders (e.g bladder pain syndrome/interstitial cystitis - BPS/IC). In a previous study we found specific mAChR expression patterns associated with BPS/IC, while correlation between protein and gene expression was lacking. Posttranslational regulatory mechanisms, e...
March 15, 2018: Acta Histochemica
Eugene Jennifer Jin, Ferdi Ridvan Kiral, Mehmet Neset Ozel, Lara Sophie Burchardt, Marc Osterland, Daniel Epstein, Heike Wolfenberg, Steffen Prohaska, Peter Robin Hiesinger
Neurons are highly polarized cells that require continuous turnover of membrane proteins at axon terminals to develop, function, and survive. Yet, it is still unclear whether membrane protein degradation requires transport back to the cell body or whether degradation also occurs locally at the axon terminal, where live observation of sorting and degradation has remained a challenge. Here, we report direct observation of two cargo-specific membrane protein degradation mechanisms at axon terminals based on a live-imaging approach in intact Drosophila brains...
March 3, 2018: Current Biology: CB
Pia Pužar Dominkuš, Matjaž Stenovec, Simona Sitar, Eva Lasič, Robert Zorec, Ana Plemenitaš, Ema Žagar, Marko Kreft, Metka Lenassi
PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells. Here, we have characterized PKH26-positive particles in lymphoblastoid B exosome samples and exosome-free controls stained by ultracentrifugation, filtration, and sucrose-cushion-based and sucrose-gradient-based procedures, using confocal imaging and asymmetric-flow field-flow fractionation coupled to multi-angle light-scattering detector analysis...
March 15, 2018: Biochimica et Biophysica Acta
Miyuki Kuno
A proton is a ubiquitous signaling ion. Many transmembrane H+ transport pathways either maintain pH homeostasis or generate acidic compartments. The osteoclast is a bone-resorbing cell, which degrades bone tissues by secreting protons and lysosomal enzymes into the resorption pit. The plasma membrane facing bone tissue (ruffled border), generated partly by fusion of lysosomes, may mimic H+ flux mechanisms regulating acidic vesicles. We identified three electrogenic H+ -fluxes in osteoclast plasma membranes-a vacuolar H+ -ATPase (V-ATPase), a voltage-gated proton channel (Hv channel) and an acid-inducible H+ -leak-whose electrophysiological profiles and regulation mechanisms differed...
March 17, 2018: Pflügers Archiv: European Journal of Physiology
Xiao-Li Xie, Jie-Tao He, Zheng-Tao Wang, Huan-Qin Xiao, Wen-Tao Zhou, Si-Hao Du, Ye Xue, Qi Wang
Methamphetamine (METH) is a widely abused psychostimulant. Lactulose is a non-absorbable sugar, which effectively decreases METH-induced neurotoxicity in rat. However, the exact mechanisms need further investigation. In this study, 5-week-old male Sprague Dawley rats received METH (15 mg/kg, 8 intraperitoneal injections, 12-hour interval) or saline and received lactulose (5.3 g/kg, oral gavage, 12-hour interval) or vehicle 2 days prior to the METH administration. Compared to the control group, in the METH alone group, cytoplasmic vacuolar degeneration in hepatocytes, higher levels of alanine transaminase, aspartate transaminase and ammonia, overproduction of reactive oxygen species (ROS) and increase of superoxide dismutase activity in the blood were observed...
March 14, 2018: Toxicology Letters
Xabier Osteikoetxea, Márton Benke, Marta Rodriguez, Krisztina Pálóczi, Barbara W Sódar, Zsuzsanna Szvicsek, Katalin Szabó-Taylor, Krisztina V Vukman, Ágnes Kittel, Zoltán Wiener, Károly Vékey, László Harsányi, Ákos Szűcs, Lilla Turiák, Edit I Buzás
AIMS: The prognosis of patients with pancreatic cancer has remained virtually unchanged with a high mortality rate compared to other types of cancers. An earlier detection would provide a time window of opportunity for treatment and prevention of deaths. In the present study we investigated extracellular vesicle (EV)-associated potential biomarkers for pancreatic cancer by directly assessing EV size-based subpopulations in pancreatic juice samples of patients with chronic pancreatitis or pancreatic cancer...
March 14, 2018: Biochemical and Biophysical Research Communications
Kenichi Morigaki, Yasushi Tanimoto
One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids...
March 14, 2018: Biochimica et Biophysica Acta
Kamilla A Mukhutdinova, Marat R Kasimov, Arthur R Giniatullin, Guzel F Zakyrjanova, Alexey M Petrov
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the initial denervation of skeletal muscle and subsequent death of motor neurons. A dying-back pattern of ALS suggests a crucial role for neuromuscular junction dysfunction. In the present study, microelectrode recording of postsynaptic currents and optical detection of synaptic vesicle traffic (FM1-43 dye) and intracellular NO levels (DAF-FM DA) were used to examine the effect of the major brain-derived cholesterol metabolite 24S-hydroxycholesterol (24S-HC, 0...
March 14, 2018: Molecular and Cellular Neurosciences
Hongling Zhu, Huizhu Gan, Zhangzhen Shi, Yu Pei, Butong Sun
No abstract text is available yet for this article.
February 22, 2018: Clinical Genitourinary Cancer
Brianne J Lindsay, Michal M Bonar, Ian N Costas-Cancelas, Kristin Hunt, Alexander I Makarkov, Sabrina Chierzi, Connie M Krawczyk, Nathalie Landry, Brian J Ward, Isabelle Rouiller
Plant-made virus-like particle (VLP) vaccines that display wild-type influenza hemagglutinin (HA) are rapidly advancing through clinical trials. Produced by transient transfection of Nicotiana benthamiana, these novel vaccines are unusually immunogenic, eliciting both humoral and cellular responses. Here, we directly visualized VLPs bearing either HA trimers derived from strains A/California/7/2009 or A/Indonesia/5/05 using cryo-electron microscopy and determined the 3D organization of the VLPs using cryo-electron tomography...
March 14, 2018: Vaccine
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