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https://www.readbyqxmd.com/read/28748462/in-vivo-rat-t-lymphocyte-pig-a-assay-detection-and-expansion-of-cells-deficient-in-the-gpi-anchored-cd48-surface-marker-for-analysis-of-mutation-in-the-endogenous-pig-a-gene
#1
Vasily N Dobrovolsky, Javier Revollo, Dayton M Petibone, Robert H Heflich
The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28748272/a-small-cellulose-binding-domain-protein-cbd1-in-phytophthora-is-highly-variable-in-the-non-binding-amino-terminus
#2
Richard W Jones, Frances G Perez
The small cellulose-binding-domain protein CBD1 is tightly bound to the cellulosic cell wall of the plant pathogenic stramenopile Phytophthora infestans. Transgene expression of the protein in potato plants also demonstrated binding to plant cell walls. A study was undertaken using 47 isolates of P. infestans from a worldwide collection, along with 17 other Phytophthora species and a related pathogen Plasmopara halstedii, to determine if the critical cell wall protein is subject to amino acid variability. Within the amino acid sequence of the secreted portion of CBD 1, encoded by the P...
July 26, 2017: Current Microbiology
https://www.readbyqxmd.com/read/28747497/the-genetic-stability-of-piv5-vectored-rsv-vaccine-candidates-after-in-vitro-and-in-vivo-passage
#3
Shannon I Phan, Carolyn M Adam, Zhenhai Chen, Michael Citron, Xiaoping Liang, Amy S Espeseth, Dai Wang, Biao He
Human respiratory syncytial virus (RSV) is the leading etiologic agent of lower respiratory tract infections in children, but no licensed vaccine exists. Previously, our laboratory developed two parainfluenza virus 5 (PIV5)-based RSV vaccine candidates that protect mice against RSV challenge. PIV5 was engineered to express either the RSV fusion protein (F) or the RSV major attachment glycoprotein (G) between the HN and L genes of the PIV5 genome [PIV5-RSV-F (HN-L) or PIV5-RSV-G (HN-L)]. To investigate the stability of the vaccine candidates in vitro, they were passaged in Vero cells at high and low multiplicities of infection (MOI) for eleven generations and the genome sequences, growth kinetics, and protein expression of the resulting viruses were compared with those of the parent viruses...
July 26, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28747472/disentangling-sources-of-variation-in-ssu-rdna-sequences-from-single-cell-analyses-of-ciliates-impact-of-copy-number-variation-and-experimental-error
#4
Chundi Wang, Tengteng Zhang, Yurui Wang, Laura A Katz, Feng Gao, Weibo Song
Small subunit ribosomal DNA (SSU rDNA) is widely used for phylogenetic inference, barcoding and other taxonomy-based analyses. Recent studies indicate that SSU rDNA of ciliates may have a high level of sequence variation within a single cell, which impacts the interpretation of rDNA-based surveys. However, sequence variation can come from a variety of sources including experimental errors, especially the mutations generated by DNA polymerase in PCR. In the present study, we explore the impact of four DNA polymerases on sequence variation and find that low-fidelity polymerases exaggerate the estimates of single-cell sequence variation...
July 26, 2017: Proceedings. Biological Sciences
https://www.readbyqxmd.com/read/28747214/culturing-and-transcriptome-profiling-of-progenitor-like-colonies-derived-from-adult-mouse-pancreas
#5
Dongshen Ma, Shanshan Tang, Jing Song, Qiong Wu, Fangfang Zhang, Yun Xing, Yi Pan, Yanfeng Zhang, Jingwei Jiang, Yubin Zhang, Liang Jin
BACKGROUND: Transplantation of insulin-producing cells is considered an important diabetes therapy. Many research studies have shown that insulin-producing cells can be derived from the in-vitro cultured pancreatic colonies with self-renewal ability and multilineage potential. Even though these progenitor-like colonies have been prepared from adult pancreas cells, the efficient culture method is hardly established and regulation of the colonies is rarely known. We confirmed previously that single cells acquired from adult mouse pancreas could form cyst-like colonies in a 3D semi-solid system containing Matrigel and methylcellulose...
July 26, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/28746860/integrating-rna-sequencing-into-neuro-oncology-practice
#6
REVIEW
David S Rogawski, Nicholas A Vitanza, Angela C Gauthier, Vijay Ramaswamy, Carl Koschmann
Malignant tumors of the central nervous system (CNS) cause substantial morbidity and mortality, yet efforts to optimize chemo- and radiotherapy have largely failed to improve dismal prognoses. Over the past decade, RNA sequencing (RNA-seq) has emerged as a powerful tool to comprehensively characterize the transcriptome of CNS tumor cells in one high-throughput step, leading to improved understanding of CNS tumor biology and suggesting new routes for targeted therapies. RNA-seq has been instrumental in improving the diagnostic classification of brain tumors, characterizing oncogenic fusion genes, and shedding light on intratumor heterogeneity...
July 8, 2017: Translational Research: the Journal of Laboratory and Clinical Medicine
https://www.readbyqxmd.com/read/28746369/single-and-combined-effect-of-retinoic-acid-and-rapamycin-modulate-the-generation-activity-and-homing-potential-of-induced-human-regulatory-t-cells
#7
Enzo Candia, Paz Reyes, Camila Covian, Francisco Rodriguez, Nicolas Wainstein, Jorge Morales, Claudio Mosso, Mario Rosemblatt, Juan Alberto Fierro
Adoptive transfer of CD4+CD25+FOXP3+ regulatory T cells (Treg cells) has been successfully utilized to treat graft versus host disease and represents a promising strategy for the treatment of autoimmune diseases and transplant rejection. The aim of this study was to evaluate the effects of all-trans retinoic acid (atRA) and rapamycin (RAPA) on the number, phenotype, homing markers expression, DNA methylation, and function of induced human Treg cells in short-term cultures. Naive T cells were polyclonally stimulated and cultured for five days in the presence of different combinations of IL-2, TGF-β1, atRA and RAPA...
2017: PloS One
https://www.readbyqxmd.com/read/28746191/whole-exome-sequencing-unveils-a-frameshift-mutation-in-cngb3-for-cone-dystrophy-a-case-report-of-an-indian-family
#8
Shashank Gupta, Amit Chaurasia, Ekta Pathak, Rajeev Mishra, Vidya Nair Chaudhry, Prashaant Chaudhry, Ashim Mukherjee, Mousumi Mutsuddi
RATIONALE: Genetic elucidation of cone-dominated retinal dystrophies in Indian subcontinent is much needed to identify and catalog underlying genetic defects. In this context, the present study recruited a consanguineous Indian family affected with autosomal recessive cone dystrophy (CD). Considering the huge genetic heterogeneity and recessive inheritance of the disease, we chose to dissect out causal variant in this family by whole exome sequencing (WES). PATIENT CONCERNS: In the recruited family, three of the six siblings had complaints of poor visual acuity, photophobia, and disturbed colour vision since early childhood...
July 2017: Medicine (Baltimore)
https://www.readbyqxmd.com/read/28745859/quantitative-effects-of-o-linked-glycans-on-protein-folding
#9
Patrick K Chaffey, Xiaoyang Guan, Xinfeng Wang, Yuan Ruan, Yaohao Li, Suzannah G Miller, Amy H Tran, Theo N Koelsch, Lomax F Pass, Zhongping Tan
Protein O-glycosylation is a diverse, common, and important post-translational modification of both proteins inside the cell and those that are secreted or membrane bound. Much work has shown that O-glycosylation can alter the structure, func-tion, and physical properties of the proteins to which it is attached. One gap remaining in our understanding of O-glycoproteins is how O-glycans might affect the folding of proteins. Here, we took advantage of synthetic, homogenous O-glycopeptides to show that certain glycosylation patterns have an intrinsic effect, independent of any cellular folding machinery, on the folding pathway of a model O-glycoprotein, a carbohydrate binding module (CBM) derived from the Trichoderma reesei cellulase TrCel7A...
July 26, 2017: Biochemistry
https://www.readbyqxmd.com/read/28745433/transcriptome-analysis-in-primary-colorectal-cancer-tissues-from-patients-with-and-without-liver-metastases-using-next-generation-sequencing
#10
Sen Wang, Chuan Zhang, Zhiyuan Zhang, Wenwei Qian, Ye Sun, Bing Ji, Yue Zhang, Chunyan Zhu, Dongjian Ji, Qingyuan Wang, Yueming Sun
Colorectal cancer (CRC) is the third most common cancer worldwide and liver metastases are the leading cause of death in patients with CRC. In this study, we performed next-generation sequencing profiling on primary colorectal tumor tissues obtained from three CRC patients with liver metastases and three CRC patients without liver metastases to identify differentially expressed genes (DEGs) that might be responsible for the metastases process. After filtering 2690 DEGs, comprising 996 upregulated and 1694 downregulated RNAs, 22 upregulated and 73 downregulated DEGs were identified...
July 26, 2017: Cancer Medicine
https://www.readbyqxmd.com/read/28743584/a-single-plasmid-reverse-genetics-system-for-the-rescue-of-non-segmented-negative-strand-rna-viruses-from-cloned-full-length-cdna
#11
Ben Peeters, Olav de Leeuw
Reverse genetics systems for non-segmented negative-strand RNA viruses rely on co-transfection of a plasmid containing the full-length viral cDNA and helper plasmids encoding essential viral replication proteins. Here, a system is presented in which virus can be rescued from a single plasmid without the need for helper plasmids in cells infected with a host-restricted recombinant poxvirus that expresses T7 RNA polymerase. This approach relies on the insertion of T7 promoter sequences in the viral cDNA at positions that allow transcription of sub-genomic RNAs encoding essential viral replication proteins...
July 22, 2017: Journal of Virological Methods
https://www.readbyqxmd.com/read/28743163/alk-and-ros1-testing-on-lung-cancer-cytologic-samples-perspectives
#12
REVIEW
Pasquale Pisapia, Maria D Lozano, Elena Vigliar, Claudio Bellevicine, Francesco Pepe, Umberto Malapelle, Giancarlo Troncone
Cytologic sampling is the mainstay of diagnosing advanced lung cancer. Moreover, to select patients for personalized first-line or second-line treatment, epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) rearrangements are tested on cytologic preparations. Commercially available fluorescence in situ hybridization (FISH) and immunocytochemistry (ICC) assays have primarily been used for the identification of cells harboring ALK or ROS1 gene fusions on histologic rather than cytologic preparations...
July 25, 2017: Cancer
https://www.readbyqxmd.com/read/28742969/fabrication-of-a-new-lineage-of-artificial-luciferases-from-natural-luciferase-pools
#13
Sung Bae Kim, Ryo Nishihara, Daniel Citterio, Koji Suzuki
The fabrication of artificial luciferases (ALucs) with unique optical properties can have a fundamental impact on bioassays and molecular imaging. In this study, we developed a new lineage of ALucs with unique substrate preferences by extracting consensus amino acids from the alignment of 25 copepod luciferase sequences available in published databases. The primary sequence was first created with a sequence logo generator resulting in a total of 11 sibling sequences. Phylogenetic analysis shows that the newly fabricated ALucs form an independent branch, genetically isolated from the natural luciferases and from a prior series of ALucs produced by our laboratory using a smaller basis set...
July 25, 2017: ACS Combinatorial Science
https://www.readbyqxmd.com/read/28741097/distribution-and-phylogenetic-analysis-of-blastocystis-sp-subtypes-isolated-from-ibd-patients-and-healthy-individuals-in-iran
#14
H Mirjalali, M R Abbasi, N Naderi, Z Hasani, E S Mirsamadi, C R Stensvold, H Balaii, H Asadzadeh Aghdaei, M R Zali
Blastocystis is a single-celled intestinal parasite commonly found in humans and a broad range of animals all over the world. In humans, its role in health and disease remains unsettled. The aim of our study was to investigate the distribution of Blastocystis and Blastocystis subtypes (ST) in patients with inflammatory bowel disease (IBD) and control subjects. A total of 71 stool samples were collected from IBD patients, 69 and 2 of whom had ulcerative colitis (UC) and Crohn's Disease (CD), respectively. Moreover, 166 stool samples from healthy subjects were included as control samples...
July 25, 2017: European Journal of Clinical Microbiology & Infectious Diseases
https://www.readbyqxmd.com/read/28740759/a-positive-readout-single-transcript-reporter-for-site-specific-mrna-cleavage
#15
Nikolay Kandul, Ming Guo, Bruce A Hay
Cleavage of mRNA molecules causes their rapid degradation, thereby playing an important role in regulation of gene expression and host genome defense from viruses and transposons in bacterial and eukaryotic cells. Current negative-readout, and repressor-based positive-readout reporters of mRNA degradation have limitations. Here we report the development of a single transcript that acts as a positive reporter of mRNA cleavage. We show that placement of bacterial CopT and CopA hairpins into the 5' UTR and 3' UTR of an mRNA results in inhibition of translation of the intervening coding sequence in Drosophila...
2017: PeerJ
https://www.readbyqxmd.com/read/28739733/analysis-of-association-between-mgmt-and-p53-gene-single-nucleotide-polymorphisms-and-laryngeal-cancer
#16
Yayun Lv, Chuanliang Jia, Aihua Jiang, Hua Zhang, Yunqiang Wang, Feifei Liu, Linlin Yang, Yan Sun, Runli Lv, Xicheng Song
AIM: To investigate the p53 and O(6)-methylguanine DNA methyltransferase (MGMT)5' upstream sequence gene promoter regions for single nucleotide polymorphisms and explore the p53 gene 5' upstream sequence consisting of two haplotypes to provide a genetic marker for the incidence of laryngeal squamous cell carcinoma. MATERIALS AND METHODS: We included 96 cases of laryngeal squamous cell carcinoma and 102 controls. We used SNaPshot micro-sequencing analysis of the MGMT promoter region for four single nucleotide polymorphisms and p53 gene 5' upstream sequence loci (rs1625649, rs2287499, rs2287498, rs228749) genotypes...
August 2017: Anticancer Research
https://www.readbyqxmd.com/read/28738459/-gene-gene-interaction-on-central-obesity-in-school-aged-children-in-china
#17
L W Fu, M X Zhang, L J Wu, L W Gao, J Mi
Objective: To investigate possible effect of 6 obesity-associated SNPs in contribution to central obesity and examine whether there is an interaction in the 6 SNPs in the cause of central obesity in school-aged children in China. Methods: A total of 3 502 school-aged children who were included in Beijing Child and Adolescent Metabolic Syndrome (BCAMS) Study were selected, and based on the age and sex specific waist circumference (WC) standards in the BCAMS study, 1 196 central obese cases and 2 306 controls were identified...
July 10, 2017: Zhonghua Liu Xing Bing Xue za Zhi, Zhonghua Liuxingbingxue Zazhi
https://www.readbyqxmd.com/read/28737902/absorption-of-low-energy-uv-radiation-by-human-telomere-g-quadruplexes-generates-long-lived-guanine-radical-cations
#18
Akos Banyasz, Lara Martínez-Fernández, Clémence Balty, Marion Perron, Thierry Douki, Roberto Improta, Dimitra Markovitsi
Telomeres, which are involved in cell division, carcinogenesis, and aging and constitute important therapeutic targets, are prone to oxidative damage. This propensity has been correlated with the presence of guanine-rich sequences, capable of forming four-stranded DNA structures (G-quadruplexes). Here, we present the first study on oxidative damage of human telomere G-quadruplexes without mediation of external molecules. Our investigation has been performed for G-quadruplexes formed by folding of GGG(TTAGGG)3 single strands in buffered solutions containing Na(+) cations (TEL21/Na(+))...
July 24, 2017: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/28737741/rapidly-inducible-cas9-and-dsb-ddpcr-to-probe-editing-kinetics
#19
John C Rose, Jason J Stephany, William J Valente, Bridget M Trevillian, Ha V Dang, Jason H Bielas, Dustin J Maly, Douglas M Fowler
We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs...
July 24, 2017: Nature Methods
https://www.readbyqxmd.com/read/28737476/a-high-fidelity-method-for-genomic-sequencing-of-single-somatic-cells-reveals-a-very-high-mutational-burden
#20
Jan Vijg, Xiao Dong, Lei Zhang
Postzygotic mutations in somatic cells lead to genome mosaicism and can be the cause of cancer, possibly other human diseases and aging. Somatic mutations are difficult to detect in bulk tissue samples. Here, we review the available assays for measuring somatic mutations, with a focus on recent single-cell, whole genome sequencing methods. Impact statement Somatic mutations cause cancer, possibly other diseases and aging. Yet, very little is known about the frequency of such mutations in vivo, their distribution across the genome, and their possible functional consequences other than cancer...
July 2017: Experimental Biology and Medicine
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