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https://www.readbyqxmd.com/read/29458078/isolation-of-intact-rna-from-murine-cd4-t-cells-after-intracellular-cytokine-staining-and-fluorescence-activated-cell-sorting
#1
Shajo Kunnath-Velayudhan, Steven A Porcelli
Intracellular cytokine staining (ICS) is a powerful method for identifying functionally distinct lymphocyte subsets, and for isolating these by fluorescence activated cell sorting (FACS). Although transcriptomic analysis of cells sorted on the basis of ICS has many potential applications, this is rarely performed because of the difficulty in isolating intact RNA from cells processed using standard fixation and permeabilization buffers for ICS. To address this issue, we compared three buffers shown previously to preserve RNA in nonhematopoietic cells subjected to intracellular staining for their effects on RNA isolated from T lymphocytes processed for ICS...
February 17, 2018: Journal of Immunological Methods
https://www.readbyqxmd.com/read/29457691/apportioning-bacterial-carbon-source-utilization-in-soil-using-14-c-isotope-analysis-of-fish-targeted-bacterial-populations-sorted-by-fluorescence-activated-cell-sorting-facs-14-c-fish-facs
#2
Christos Gougoulias, Andrew Meade, Liz J Shaw
An unresolved need in microbial ecology is methodology to enable quantitative analysis of in situ microbial substrate carbon use at the population level. Here, we evaluated if a novel combination of radiocarbon- labelled substrate tracing, Fluorescence in situ Hybridisation (FISH) and Fluorescence-Activated Cell Sorting (FACS) to sort the FISH-targeted population for quantification of incorporated radioactivity (14 C-FISH-FACS) can address this need. Our test scenario used FISH probe PSE1284 targeting Pseudomonas spp...
February 19, 2018: Environmental Microbiology Reports
https://www.readbyqxmd.com/read/29457375/evidence-for-alternate-modes-of-b-cell-activation-involving-fab-acquired-n-glycosylations-in-antibody-secreting-cells-infiltrating-the-labial-salivary-glands-of-sj%C3%A3-gren-s-syndrome-patients
#3
Kristi A Koelsch, Joshua Cavett, Kenneth Smith, Jacen S Moore, Sylvain D Lehoux, Nan Jia, Tim Mather, Syed M S Quadri, Astrid Rasmussen, C Erick Kaufman, David M Lewis, Lida Radfar, Teresa A Scordino, Christopher J Lessard, Biji T Kurien, Richard D Cummings, Judith A James, Kathy L Sivils, A Darise Farris, R Hal Scofield
OBJECTIVE: To better understand the role of B cells, potential mechanisms for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's Syndrome (SS), we explored selection pressures and N-glycosylation acquired by somatic mutation (acN-glyc) in the immunoglobulin (Ig) variable regions (V-regions) of antibody secreting cells (ASCs) isolated from the minor salivary glands of SS patients and non-SS controls with sicca symptoms. METHODS: We report a novel method to produce and characterize recombinant monoclonal antibodies (mAbs) from SS patient and control labial salivary gland single-cell sorted ASC infiltrates that can be utilized to concurrently probe any other expressed genes...
February 18, 2018: Arthritis & Rheumatology
https://www.readbyqxmd.com/read/29454966/effects-of-pq-s-cytotoxicity-on-secretory-vesicles-in-astroglia-expression-alternation-of-secretogranin-ii-and-its-potential-interaction-with-intracellular-factors
#4
Xiaoni Zhan, Fengrui Li, Qiaohong Chu, Hao Pang
It has been extensively characterized that paraquat (PQ) selectively targets to the substantia nigra and exerts neurotoxic actions on dopaminergic neurons. However, a little knowledge is available about astroglia in PQ exposure, especially its complex secretory machinery. To explore this point, we built up a PQ-induced model in cultural U118 astrocyte. Since the granin family is considered as a master regulator of cargo sorting and large dense core vesicles (LDCVs) biogenesis in the regulated secretory pathway of nervous and neuroendocrine cells, the current study focused on one member, secretogranin II (SCG2) and investigated its alternation and potential relationship with other astrocyte-derived factors under PQ insult...
February 15, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29454754/mutational-analysis-of-akt1-and-pik3ca-in-intraductal-papillomas-of-the-breast-with-special-reference-to-cellular-components
#5
Chieko Mishima, Naofumi Kagara, Jun-Ichiro Ikeda, Eiichi Morii, Tomohiro Miyake, Tomonori Tanei, Yasuto Naoi, Masafumi Shimoda, Kenzo Shimazu, Seung Jin Kim, Shinzaburo Noguchi
The pathological feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti-CK19 antibody and anti-SMA antibody...
February 15, 2018: American Journal of Pathology
https://www.readbyqxmd.com/read/29454615/synthesis-and-biological-investigation-of-new-carbonic-anhydrase-ix-caix-inhibitors
#6
Kranthi Vanchanagiri, Daniel Emmerich, Monique Bruschke, Matthias Bache, Franziska Seifert, René Csuk, Dirk Vordermark, Reinhard Paschke
In this report, we describe the synthesis, characterization, in vitro anticancer activity and Carbonic anhydrase IX (CAIX) inhibition of new sulfamate conjugates of Betulin and Betulinic acid (BA). The betulinyl sulfamates were subjected to inhibit carbonic anhydrases (CA), e.g. CAIX, an attractive target for tumor-selective therapy strategies in cancer cells. Data on combined in vitro antitumor activity with CAIX inhibition are very rare. The betulinyl sulfamates were tested against five tumor cell lines and normal human skin fibroblasts...
February 15, 2018: Chemico-biological Interactions
https://www.readbyqxmd.com/read/29453607/semaphorin-7a-in-circulating-regulatory-t-cells-is-increased-in-autosomal-dominant-polycystic-kidney-disease-and-decreases-with-tolvaptan-treatment
#7
Yashang Lee, Katrina Lehmann Blount, Feng Dai, Siobhan Thompson, Jonathan Kaufman Scher, Sherrie Bitterman, Madeline Droher, Erica L Herzog, Gilbert Moeckel, Anil Karihaloo, Neera K Dahl
BACKGROUND: Semaphorin 7A (SEMA7A) is an immunomodulating protein implicated in lung and liver fibrosis. In autosomal-dominant polycystic kidney disease (ADPKD), the progressive expansion of renal cysts, inflammation, and subsequent renal fibrosis leads to end-stage renal disease (ESRD). SEMA7A may play a role in renal fibrosis and in ADPKD. METHODS: We evaluated Sema7a in a mouse model of renal fibrosis and determined the expression of SEMA7A in human ADPKD kidney...
February 16, 2018: Clinical and Experimental Nephrology
https://www.readbyqxmd.com/read/29453057/mammarenaviruses-deleted-from-their-z-gene-are-replicative-and-produce-an-infectious-progeny-in-bhk-21-cells
#8
Amélie D Zaza, Cécile H Herbreteau, Christophe N Peyrefitte, Sébastien F Emonet
Mammarenaviruses bud out of infected cells via the recruitment of the endosomal sorting complex required for transport through late domain motifs localized into their Z protein. Here, we demonstrated that mammarenaviruses lacking this protein can be rescued and are replicative, despite a 3-log reduction in virion production, in BHK-21 cells, but not in five other cell lines. Mutations of putative late domain motifs identified into the viral nucleoprotein resulted in the almost complete abolition of infectious virion production by Z-deleted mammarenaviruses...
February 13, 2018: Virology
https://www.readbyqxmd.com/read/29452515/intelligent-anticancer-drug-delivery-performances-of-two-poly-n-isopropylacrylamide-based-magnetite-nanohydrogels
#9
Nahid Poorgholy, Bakhshali Massoumi, Marjan Ghorbani, Mehdi Jaymand, Hamed Hamishehkar
This article evaluates the anticancer drug delivery performances of two nanohydrogels composed of poly(N-isopropylacrylamide-co-itaconic anhydride) [P(NIPAAm-co-IA)], poly(ethylene glycol) (PEG), and Fe 3 O 4 nanoparticles. For this purpose, the magnetite nanohydrogels (MNHGs) were loaded with doxorubicin hydrochloride (DOX) as a universal anticancer drug. The morphologies and magnetic properties of the DOX-loaded MNHGs were investigated using transmission electron microscopy (TEM) and vibrating-sample magnetometer (VSM), respectively...
February 16, 2018: Drug Development and Industrial Pharmacy
https://www.readbyqxmd.com/read/29452406/negative-association-between-sclerostin-and-insl3-in-isolated-human-osteocytes-and-in-klinefelter-syndrome-new-hints-for-testis-bone-crosstalk
#10
Andrea Di Nisio, Luca De Toni, Maria Santa Rocca, Marco Ghezzi, Riccardo Selice, Giuseppe Taglialavoro, Alberto Ferlin, Carlo Foresta
Context/Objective: The regulation of bone mass by the testis is a well-recognized mechanism, but the role of Leydig-specific marker insuline-like 3 peptide (INSL3) on the most abundant bone cell population, osteocytes, is unknown. In this study we aimed to investigate the relationship between INSL3 and Sclerostin, an osteocyte-specific protein that negatively regulates bone formation. Design/Setting: Serum sclerostin and INSL3 levels were evaluated in Klinefelter Syndrome (KS) and healthy controls...
February 14, 2018: Journal of Clinical Endocrinology and Metabolism
https://www.readbyqxmd.com/read/29452354/alpha-synuclein-inhibits-snx3-retromer-mediated-retrograde-recycling-of-iron-transporters-in-s-cerevisiae-and-c-elegans-models-of-parkinson-s-disease
#11
Dhaval Patel, Chuan Xu, Sureshbabu Nagarajan, Zhengchang Liu, Wayne O Hemphill, Runhua Shi, Vladimir N Uversky, Guy A Caldwell, Kim A Caldwell, Stephan N Witt
We probed the role of alpha-synuclein (α-syn) in modulating sorting nexin 3 (Snx3)-retromer-mediated recycling of iron transporters in Saccharomyces cerevisiae and Caenorhabditis elegans. In yeast, the membrane-bound heterodimer Fet3/Ftr1 is the high affinity iron importer. Fet3 is a membrane-bound multicopper ferroxidase, whose ferroxidase domain is orthologous to human ceruloplasmin (Cp), that oxidizes external Fe+2 to Fe+3; the Fe+3 ions then channel through the Ftr1 permease into the cell. When the concentration of external iron is low (< 1 µM), Fet3/Ftr1 is maintained on the plasma membrane by retrograde endocytic-recycling; whereas, when the concentration of external iron is high (> 10 µM), Fet3/Ftr1 is endocytosed and shunted to the vacuole for degradation...
February 14, 2018: Human Molecular Genetics
https://www.readbyqxmd.com/read/29449449/conserved-and-divergent-features-of-mesenchymal-progenitor-cell-types-within-the-cortical-nephrogenic-niche-of-the-human-and-mouse-kidney
#12
Nils O Lindström, Jinjin Guo, Albert D Kim, Tracy Tran, Qiuyu Guo, Guilherme De Sena Brandine, Andrew Ransick, Riana K Parvez, Matthew E Thornton, Laurence Basking, Brendan Grubbs, Jill A McMahon, Andrew D Smith, Andrew P McMahon
Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2 + nephron progenitor cells (NPCs) and Foxd1 + interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney...
February 15, 2018: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/29449377/haploid-embryonic-stem-cells-can-be-enriched-and-maintained-by-simple-filtration
#13
Chao Qu, Meng Yan, Suming Yang, Lingbo Wang, Qi Yin, Yuan Liu, Yeguang Chen, Jinsong Li
Haploid mammalian embryonic stem cells (haESCs) serve as a powerful tool for genetic analyses at both cellular and organismal levels. However, spontaneous diploidization of haESCs limits their use in these analyses. Addition of small molecules to culture medium to control the cell cycle can slow down diploidization, but cell-sorting methods such as FACS are still required to enrich haploid cells for long-term maintenance in vitro. Here, acting on our observation that haploid and diploidized cells differ in diameter, we developed a simplified filtration method to enrich haploid cells from cultured haESCs...
February 15, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29445238/vps35-depletion-does-not-impair-presynaptic-structure-and-function
#14
Sonia Vazquez-Sanchez, Sander Bobeldijk, Marien P Dekker, Linda van Keimpema, Jan R T van Weering
The endosomal system is proposed as a mediator of synaptic vesicle recycling, but the molecular recycling mechanism remains largely unknown. Retromer is a key protein complex which mediates endosomal recycling in eukaryotic cells, including neurons. Retromer is important for brain function and mutations in retromer genes are linked to neurodegenerative diseases. In this study, we aimed to determine the role of retromer in presynaptic structure and function. We assessed the role of retromer by knocking down VPS35, the core subunit of retromer, in primary hippocampal mouse neurons...
February 14, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29445159/microfluidic-cell-sorting-by-stiffness-to-examine-heterogenic-responses-of-cancer-cells-to-chemotherapy
#15
Muhymin Islam, Roman Mezencev, Brynn McFarland, Hannah Brink, Betsy Campbell, Bushra Tasadduq, Edmund K Waller, Wilbur Lam, Alexander Alexeev, Todd Sulchek
Cancers consist of a heterogeneous populations of cells that may respond differently to treatment through drug-resistant sub-populations. The scarcity of these resistant sub-populations makes it challenging to understand how to counter their resistance. We report a label-free microfluidic approach to separate cancer cells treated with chemotherapy into sub-populations enriched in chemoresistant and chemosensitive cells based on the differences in cellular stiffness. The sorting approach enabled analysis of the molecular distinctions between resistant and sensitive cells...
February 14, 2018: Cell Death & Disease
https://www.readbyqxmd.com/read/29444958/cdc48-and-ubiquilins-confer-selective-anterograde-protein-sorting-and-entry-into-the-multivesicular-body-in-yeast
#16
Rachel Kama, Galina Gabriely, Vydehi Kanneganti, Jeffrey E Gerst
Cdc48/p97 is known primarily for the retro-translocation of misfolded proteins in ER-associated protein degradation (ERAD). Here, we uncover a novel function for both Cdc48 and the conserved UBA-UBL ubiquitin receptor (ubiquilin) proteins in yeast ( e.g. Ddi1, Dsk2, Rad23), which deliver ubiquitinated proteins to the proteasome for degradation. We show that Cdc48, its core adaptors Npl4 and Ufd1, and the ubiquilins confer the constitutive anterograde delivery of carboxypeptidase S (Cps1), a membranal hydrolase, to the multivesicular body (MVB) and vacuolar lumen...
February 14, 2018: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/29444383/stemness-and-anti-cancer-drug-resistance-in-abcg2-highly-expressed-pancreatic-cancer-is-induced-on-3d-culture-condition
#17
Norihiko Sasaki, Toshiyuki Ishiwata, Fumio Hasegawa, Masaki Michishita, Hiroki Kawai, Yoko Matsuda, Tomio Arai, Naoshi Ishikawa, Junko Aida, Kaiyo Takubo, Masashi Toyoda
The expression of ATP-binding cassette subfamily G member 2 (ABCG2) is related with tumorigenic cancer stem cells (CSCs) in several cancers. However, the effects of ABCG2 on CSC-related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2-) and high (ABCG2+)-ABCG2-expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC-1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2- cells...
February 14, 2018: Cancer Science
https://www.readbyqxmd.com/read/29443872/ap-1-kif13a-blocking-peptides-impair-melanosome-maturation-and-melanin-synthesis
#18
Cécile Campagne, Léa Ripoll, Floriane Gilles-Marsens, Graça Raposo, Cédric Delevoye
Melanocytes are specialized cells that generate unique organelles called melanosomes in which melanin is synthesized and stored. Melanosome biogenesis and melanocyte pigmentation require the transport and delivery of melanin synthesizing enzymes, such as tyrosinase and related proteins (e.g., TYRP1), from endosomes to maturing melanosomes. Among the proteins controlling endosome-melanosome transport, AP-1 together with KIF13A coordinates the endosomal sorting and trafficking of TYRP1 to melanosomes. We identify here β1-adaptin AP-1 subunit-derived peptides of 5 amino acids that block the interaction of KIF13A with AP-1 in cells...
February 14, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29443113/simple-generation-of-a-high-yield-culture-of-induced-neurons-from-human-adult-skin-fibroblasts
#19
Shelby Shrigley, Karolina Pircs, Roger A Barker, Malin Parmar, Janelle Drouin-Ouellet
Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process...
February 5, 2018: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29440677/nanobody-triggered-lockdown-of-vsrs-reveals-ligand-reloading-in-the-golgi
#20
Simone Früholz, Florian Fäßler, Üner Kolukisaoglu, Peter Pimpl
Protein degradation in lytic compartments is crucial for eukaryotic cells. At the heart of this process, vacuolar sorting receptors (VSRs) bind soluble hydrolases in the secretory pathway and release them into the vacuolar route. Sorting efficiency is suggested to result from receptor recycling. However, how and to where plant VSRs recycle remains controversial. Here we present a nanobody-epitope interaction-based protein labeling and tracking approach to dissect their anterograde and retrograde transport routes in vivo...
February 13, 2018: Nature Communications
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