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https://www.readbyqxmd.com/read/28922845/pot1-mediated-%C3%AE-integration-strategy-for-high-copy-stable-expression-of-heterologous-proteins-in-saccharomyces-cerevisiae
#1
Xiaofei Song, Quanli Liu, Jiwei Mao, Yuzhen Wu, Yuanzi Li, Kai Gao, Xiuming Zhang, Yanling Bai, Haijin Xu, Mingqiang Qiao
In biotechnological industry, increased expression cassette stability and copy number serve as important means of maintaining consistently high production levels of heterologous proteins in Saccharomyces cerevisiae. In this study, we combined δ sequences for site-specific integration with TPI1 gene from Schizosaccharomyces pombe (POT1) as a selection marker to realize high-copy integration and stable expression of heterologous proteins in S. cerevisiae. With the newly developed POT1 platform, a 32-copy integration of enhanced green fluorescent protein (EGFP) expression cassette was obtained in a single round and was stably maintained after 100 generations of growth in a rich complex medium...
September 1, 2017: FEMS Yeast Research
https://www.readbyqxmd.com/read/28922540/ape1-ref-1-knockdown-in-pancreatic-ductal-adenocarcinoma-characterizing-gene-expression-changes-and-identifying-novel-pathways-using-single-cell-rna-sequencing
#2
Fenil Shah, Emery Goossens, Nadia M Atallah, Michelle Grimard, Mark R Kelley, Melissa L Fishel
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer-related pathways. Because APE1 is essential for cell viability, generation of APE1 knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single-cell RNA Sequencing to identify differentially expressed genes in relation to APE1 protein levels within the cell...
September 18, 2017: Molecular Oncology
https://www.readbyqxmd.com/read/28919852/dendritic-properties-control-energy-efficiency-of-action-potentials-in-cortical-pyramidal-cells
#3
Guosheng Yi, Jiang Wang, Xile Wei, Bin Deng
Neural computation is performed by transforming input signals into sequences of action potentials (APs), which is metabolically expensive and limited by the energy available to the brain. The metabolic efficiency of single AP has important consequences for the computational power of the cell, which is determined by its biophysical properties and morphologies. Here we adopt biophysically-based two-compartment models to investigate how dendrites affect energy efficiency of APs in cortical pyramidal neurons. We measure the Na(+) entry during the spike and examine how it is efficiently used for generating AP depolarization...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28919131/optogenetic-targeting-of-cardiac-myocytes-and-non-myocytes-tools-challenges-and-utility
#4
Callum M Johnston, Eva A Rog-Zielinska, Eike M Wülfers, Torsten Houwaart, Urszula Siedlecka, Thomas Knöpfel, Peter Kohl, Franziska Schneider-Warme
In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes...
September 15, 2017: Progress in Biophysics and Molecular Biology
https://www.readbyqxmd.com/read/28917036/selenocysteine-trna-ser-sec-the-central-component-of-selenoprotein-biosynthesis-isolation-identification-modification-and-sequencing
#5
Bradley A Carlson, Byeong Jae Lee, Petra A Tsuji, Paul R Copeland, Ulrich Schweizer, Vadim N Gladyshev, Dolph L Hatfield
The selenocysteine (Sec) tRNA([Ser]Sec) population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA([Ser]Sec) population by one or more procedures, and finally resolving the two isoforms from each other...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28916515/reversing-thyroid-hormone-mediated-repression-of-a-hsv-1-promoter-via-computationally-guided-mutagenesis
#6
Robert W Figliozzi, Feng Chen, Shaochung V Hsia
Thyroid hormones (TH or T3) and their DNA binding nuclear receptors (TRs), direct transcriptional regulation in diverse ways depending on the host cell environment and specific promoter characteristics of TH sensitive genes. This study sought to elucidate the impact on transcriptional repression of nucleotide sequence/orientation within TR binding sites, TR elements, (TREs) of TH sensitive promoters, to better understand ligand dependent transcriptional repression of wild-type promoters. Computational analysis of the HSV-1 thymidine kinase (TK) gene TRE bound by TR and RXR revealed a single TRE point mutation sufficient to reverse the TRE orientation...
September 15, 2017: Journal of Cell Science
https://www.readbyqxmd.com/read/28915574/comparative-study-of-whole-genome-amplification-and-next-generation-sequencing-performance-of-single-cancer-cells
#7
Anna Babayan, Malik Alawi, Michael Gormley, Volkmar Müller, Harriet Wikman, Ryan P McMullin, Denis A Smirnov, Weimin Li, Maria Geffken, Klaus Pantel, Simon A Joosse
BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation...
August 22, 2017: Oncotarget
https://www.readbyqxmd.com/read/28915495/impact-of-chemotherapy-on-metabolic-reprogramming-characterization-of-the-metabolic-profile-of-breast-cancer-mda-mb-231-cells-using-1-h-hr-mas-nmr-spectroscopy
#8
Roberta M Maria, Wanessa F Altei, Heloisa S Selistre-de-Araujo, Luiz A Colnago
Doxorubicin, cisplatin, and tamoxifen are part of many chemotherapeutic regimens. However, studies investigating the effect of chemotherapy on the metabolism of breast cancer cells are still limited. We used (1)H high-resolution magic angle spinning (HR-MAS) NMR spectroscopy to study the metabolic profile of human breast cancer MDA-MB-231 cells either untreated (control) or treated with tamoxifen, cisplatin, and doxorubicin. (1)H HR-MAS NMR single pulse spectra evidenced signals from all mobile cell compounds, including fatty acids (membranes), water-soluble proteins, and metabolites...
September 12, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28915270/the-single-cyclic-nucleotide-specific-phosphodiesterase-of-the-intestinal-parasite-giardia-lamblia-represents-a-potential-drug-target
#9
Stefan Kunz, Vreni Balmer, Geert Jan Sterk, Michael P Pollastri, Rob Leurs, Norbert Müller, Andrew Hemphill, Cornelia Spycher
BACKGROUND: Giardiasis is an intestinal infection correlated with poverty and poor drinking water quality, and treatment options are limited. According to the Center for Disease Control and Prevention, Giardia infections afflict nearly 33% of people in developing countries, and 2% of the adult population in the developed world. This study describes the single cyclic nucleotide-specific phosphodiesterase (PDE) of G. lamblia and assesses PDE inhibitors as a new generation of anti-giardial drugs...
September 15, 2017: PLoS Neglected Tropical Diseases
https://www.readbyqxmd.com/read/28914635/clinical-genetics-of-craniosynostosis
#10
Andrew O M Wilkie, David Johnson, Steven A Wall
PURPOSE OF REVIEW: When providing accurate clinical diagnosis and genetic counseling in craniosynostosis, the challenge is heightened by knowledge that etiology in any individual case may be entirely genetic, entirely environmental, or anything in between. This review will scope out how recent genetic discoveries from next-generation sequencing have impacted on the clinical genetic evaluation of craniosynostosis. RECENT FINDINGS: Survey of a 13-year birth cohort of patients treated at a single craniofacial unit demonstrates that a genetic cause of craniosynostosis can be identified in one quarter of cases...
September 14, 2017: Current Opinion in Pediatrics
https://www.readbyqxmd.com/read/28913769/plantactinospora-solaniradicis-sp-nov-a-novel-actinomycete-isolated-from-the-root-of-a-tomato-plant-solanum-lycopersicum-l
#11
Wenchao Li, Xiaowei Guo, Linlin Shi, Junwei Zhao, Liangliang Yan, Xiaotong Zhong, Chen Zhang, Yufei Chen, Xiangjing Wang, Wensheng Xiang
A Gram-positive, non-motile actinomycete, designated strain NEAU-FJL1(T), was isolated from tomato root (Solanum lycopersicum L.) collected from Harbin, Heilongjiang province, north China. The strain formed single spores with smooth surfaces from substrate mycelia. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NEAU-FJL1(T) should be affiliated with the genus Plantactinospora and forms a distinct branch with its close neighbour Plantactinospora soyae NEAU-gxj3(T) (99.2% sequence similarity)...
September 14, 2017: Antonie Van Leeuwenhoek
https://www.readbyqxmd.com/read/28912603/novel-insight-into-streptozotocin-induced-diabetic-rats-from-the-protein-misfolding-perspective
#12
Edgar Leyva-García, Reyna Lara-Martínez, Liborio Morán-Zanabria, Cristina Revilla-Monsalve, Luis Felipe Jiménez-García, Norma Oviedo, Chiharu Murata, Eulalia Garrido-Magaña, Nelly F Altamirano-Bustamante, Myriam M Altamirano-Bustamante
Protein folding is a process of self-assembly defined by the sequence of the amino acids of the protein involved. Additionally, proteins tend to unfold, misfold and aggregate due to both intrinsic and extrinsic causes. Human islet amyloid polypeptide (hIAPP) aggregation is an early step in diabetes mellitus. However, the aggregation of rat IAPP (rIAPP) remains an open question. Adult female Sprague-Dawley rats weighing 150-250 g were divided into two groups. The experimental group (streptozotocin [STZ]) (n = 21) received an intraperitoneal injection of a single dose of 40 mg/kg STZ...
September 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28912486/the-homologous-recombination-machinery-orchestrates-post-replication-dna-repair-during-self-renewal-of-mouse-embryonic-stem-cells
#13
Eui-Hwan Choi, Seobin Yoon, Kyung-Soon Park, Keun P Kim
Embryonic stem (ES) cells require homologous recombination (HR) to cope with genomic instability caused during self-renewal. Here, we report expression dynamics and localization of endogenous HR factors in DNA break repair of ES cells. In addition, we analyzed gene expression patterns of HR-related factors at the transcript level with RNA-sequencing experiments. We showed that ES cells constitutively expressed diverse HR proteins throughout the cell cycle and that HR protein expression was not significantly changed even in the DNA damaging conditions...
September 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28912341/the-role-of-blm-helicase-in-homologous-recombination-gene-conversion-tract-length-and-recombination-between-diverged-sequences-in-drosophila
#14
Henry A Ertl, Daniel P Russo, Noori Srivastava, Joseph T Brooks, Thu N Dao, Jeannine R LaRocque
DNA double-strand breaks (DSBs) are a particularly deleterious class of DNA damage that threatens genome integrity. DSBs are repaired by three pathways: non-homologous end joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). Drosophila melanogaster Blm (DmBlm) is the ortholog of Saccharomyces cerevisiae SGS1 and human BLM, and has been shown to suppress crossovers in mitotic cells and repair mitotic DNA gaps via HR. To further elucidate the role of DmBlm in repair of a simple DSB, and in particular recombination mechanisms, we utilized the DR-white and DR-white...
September 14, 2017: Genetics
https://www.readbyqxmd.com/read/28911805/delivery-strategies-of-the-crispr-cas9-gene-editing-system-for-therapeutic-applications
#15
REVIEW
Chang Liu, Li Zhang, Hao Liu, Kun Cheng
The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications...
September 11, 2017: Journal of Controlled Release: Official Journal of the Controlled Release Society
https://www.readbyqxmd.com/read/28911117/structure-based-domain-assignment-in-leishmania-infantum-endog-characterization-of-a-ph-dependent-regulatory-switch-and-a-c-terminal-extension-that-largely-dictates-dna-substrate-preferences
#16
Cristina Oliva, Pedro A Sánchez-Murcia, Eva Rico, Ana Bravo, Margarita Menéndez, Federico Gago, Antonio Jiménez-Ruiz
Mitochondrial endonuclease G from Leishmania infantum (LiEndoG) participates in the degradation of double-stranded DNA (dsDNA) during parasite cell death and is catalytically inactive at a pH of 8.0 or above. The presence, in the primary sequence, of an acidic amino acid-rich insertion exclusive to trypanosomatids and its spatial position in a homology-built model of LiEndoG led us to postulate that this peptide stretch might act as a pH sensor for self-inhibition. We found that a LiEndoG variant lacking residues 145-180 is indeed far more active than its wild-type counterpart at pH values >7...
September 6, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28911038/spliceman2-a-computational-web-server-that-predicts-defects-in-pre-mrna-splicing
#17
Kamil Jan Cygan, Clayton Hendrick Sanford, William Guy Fairbrother
Summary: Most pre-mRNA transcripts in eukaryotic cells must undergo splicing to remove introns and join exons, and splicing elements present a large mutational target for disease-causing mutations. Splicing elements are strongly position dependent with respect to the transcript annotations. In 2012, we presented Spliceman, an online tool that used positional dependence to predict how likely distant mutations around annotated splice sites were to disrupt splicing. Here, we present an improved version of the previous tool that will be more useful for predicting the likelihood of splicing mutations...
September 15, 2017: Bioinformatics
https://www.readbyqxmd.com/read/28910279/longitudinal-multiparameter-single-cell-analysis-of-macaques-immunized-with-pneumococcal-protein-conjugated-or-unconjugated-polysaccharide-vaccines-reveals-distinct-antigen-specific-memory-b-cell-repertoires
#18
Bin Jia, Lisa K McNeil, Christopher D Dupont, Konstantinos Tsioris, Rachel M Barry, Ingrid L Scully, Adebola O Ogunniyi, Christopher Gonzalez, Michael W Pride, Todd M Gierahn, Paul A Liberator, Kathrin U Jansen, J Christopher Love
BACKGROUND: The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to Streptococcus pneumoniae. METHODS: We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC) or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS) using microengraving (a single-cell analysis method) and single-cell RT-PCR...
2017: PloS One
https://www.readbyqxmd.com/read/28905511/talen-mediated-targeted-mutagenesis-of-more-than-100-comt-copies-alleles-in-highly-polyploid-sugarcane-improves-saccharification-efficiency-without-compromising-biomass-yield
#19
Baskaran Kannan, Je Hyeong Jung, Geoffrey W Moxley, Sun-Mi Lee, Fredy Altpeter
Sugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first and second generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose as well as cell wall bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O-methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass...
September 14, 2017: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/28904743/complete-genome-sequence-of-the-sulfur-oxidizing-chemolithoautotrophic-sulfurovum-lithotrophicum-42bkt-t
#20
Wooyoung Jeon, Lia Priscilla, Gyuyeon Park, Heeseok Lee, Narae Lee, Dongyup Lee, Hyuksung Kwon, Iksung Ahn, Changha Lee, Hongweon Lee, Jungoh Ahn
A sulfur-oxidizing chemolithoautotrophic bacterium, Sulfurovum lithotrophicum 42BKT(T), isolated from hydrothermal sediments in Okinawa, Japan, has been used industrially for CO2 bio-mitigation owing to its ability to convert CO2 into C5H8NO4(-) at a high rate of specific mitigation (0.42 g CO2/cell/h). The genome of S. lithotrophicum 42BKT(T) comprised of a single chromosome of 2217,891 bp with 2217 genes, including 2146 protein-coding genes and 54 RNA genes. Here, we present its complete genome-sequence information, including information about the genes encoding enzymes involved in CO2 fixation and sulfur oxidation...
2017: Standards in Genomic Sciences
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