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https://www.readbyqxmd.com/read/29150665/a-streamlined-cloning-workflow-minimising-the-time-to-strain-pipeline-for-pichia-pastoris
#1
Kate E Royle, Karen Polizzi
Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs...
November 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29150512/a-genetic-system-for-the-thermophilic-acetogenic-bacterium-thermoanaerobacter-kivui
#2
Mirko Basen, Irina Geiger, Laura Henke, Volker Müller
Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H2 + CO2 or with CO, producing acetate as major product. While a genome-derived model of acetogenesis has been developed, only few physiological or biochemical experiments towards the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted, markerless gene deletions and for integration of genes into the genome of T...
November 17, 2017: Applied and Environmental Microbiology
https://www.readbyqxmd.com/read/29150435/biochemical-characterization-of-ints3-and-c9orf80-two-subunits-of-hnabp1-2-heterotrimeric-complex-in-nucleic-acid-binding
#3
Venkatasubramanian Vidhyasagar, Yujiong He, Manhong Guo, Tanu Talwar, Ravi Shankar Singh, Manisha Yadav, George Katselis, Franco J Vizeacoumar, Erique Lukong, Yuliang Wu
Human Nucleic Acid Binding Protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single stranded (ss) DNA and RNA binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In this study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution, however C9ORF80 exhibits anomalous behavior on SDS-PAGE and gel filtration because of 48% random coil present in the protein...
November 17, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/29150381/a-plea-for-the-renewal-of-the-isbr
#4
Giovanni Tagliabue, Marcel Kuntz, Henry I Miller, Klaus Ammann
The recent meeting of the International Society for Biosafety Research (ISBR) focused on so-called genetically modified organisms. For decades, in most regulatory frameworks, recombinant DNA-modified organisms have been the wrong focus of unbalanced agri-food regulations. The ISBR should instead adopt a scientifically defensible and truly risk-based perspective, abandoning a misleading pseudo-category.
November 14, 2017: Trends in Biotechnology
https://www.readbyqxmd.com/read/29149984/corneal-xenotransplantation-from-pig-to-rhesus-monkey-no-signs-of-transmission-of-endogenous-porcine-retroviruses
#5
A Li, Y Zhang, Y Liu, Z Pan
BACKGROUND: Xenotransplantations of pig corneas have become an attractive alternative to human corneas. Such xenotransplantations carry the danger, however, of transmission of porcine endogenous retroviruses (PERVs). Here, we investigated whether porcine corneas harbor viral DNA and RNA and whether transplantation to a nonhuman primate would lead to host PERV infection. METHODS: Monkey vein endothelial cells (MVECs) were inoculated with porcine aortic endothelial cell (PAEC) supernatants, and DNA and total RNA of MVECs were tested for PERV by polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) assays...
November 2017: Transplantation Proceedings
https://www.readbyqxmd.com/read/29149457/hmgb1-controls-liver-cancer-initiation-through-yap-dependent-aerobic-glycolysis
#6
Ruochan Chen, Shan Zhu, Xue-Gong Fan, Haichao Wang, Michael T Lotze, Herbert J Zeh, Timothy R Billiar, Rui Kang, Daolin Tang
Emerging studies have suggested that the Hippo pathway is involved in the tumorigenesis of hepatocellular carcinoma (HCC). However, the key regulator of the Hippo pathway in liver tumor metabolic reprogramming remains elusive. Here, we provide evidence to support that high mobility group box 1 (HMGB1), a chromosomal protein, plays a role in the regulation of the Hippo pathway during liver tumorigenesis. Cre/loxP recombination-mediated HMGB1 depletion in hepatocytes blocks diethylnitrosamine-induced liver cancer initiation in mice, whereas shRNA-mediated gene silencing of HMGB1 inhibits HCC cell proliferation...
November 17, 2017: Hepatology: Official Journal of the American Association for the Study of Liver Diseases
https://www.readbyqxmd.com/read/29149197/sieve-analysis-of-breakthrough-hiv-1-sequences-in-hvtn-505-identifies-vaccine-pressure-targeting-the-cd4-binding-site-of-env-gp120
#7
Allan C deCamp, Morgane Rolland, Paul T Edlefsen, Eric Sanders-Buell, Breana Hall, Craig A Magaret, Andrew J Fiore-Gartland, Michal Juraska, Lindsay N Carpp, Shelly T Karuna, Meera Bose, Steven LePore, Shana Miller, Annemarie O'Sullivan, Kultida Poltavee, Hongjun Bai, Kalpana Dommaraju, Hong Zhao, Kim Wong, Lennie Chen, Hasan Ahmed, Derrick Goodman, Matthew Z Tay, Raphael Gottardo, Richard A Koup, Robert Bailer, John R Mascola, Barney S Graham, Mario Roederer, Robert J O'Connell, Nelson L Michael, Merlin L Robb, Elizabeth Adams, Patricia D'Souza, James Kublin, Lawrence Corey, Daniel E Geraghty, Nicole Frahm, Georgia D Tomaras, M Juliana McElrath, Lisa Frenkel, Sheila Styrchak, Sodsai Tovanabutra, Magdalena E Sobieszczyk, Scott M Hammer, Jerome H Kim, James I Mullins, Peter B Gilbert
Although the HVTN 505 DNA/recombinant adenovirus type 5 vector HIV-1 vaccine trial showed no overall efficacy, analysis of breakthrough HIV-1 sequences in participants can help determine whether vaccine-induced immune responses impacted viruses that caused infection. We analyzed 480 HIV-1 genomes sampled from 27 vaccine and 20 placebo recipients and found that intra-host HIV-1 diversity was significantly lower in vaccine recipients (P ≤ 0.04, Q-values ≤ 0.09) in Gag, Pol, Vif and envelope glycoprotein gp120 (Env-gp120)...
2017: PloS One
https://www.readbyqxmd.com/read/29147784/molecular-characterization-of-faba-bean-necrotic-yellows-viruses-in-tunisia
#8
Simona Kraberger, Safaa G Kumari, Asma Najar, Daisy Stainton, Darren P Martin, Arvind Varsani
Faba bean necrotic yellows virus (FBNYV) (genus Nanovirus; family Nanoviridae) has a genome comprising eight individually encapsidated circular single-stranded DNA components. It has frequently been found infecting faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) in association with satellite molecules (alphasatellites). Genome sequences of FBNYV from Azerbaijan, Egypt, Iran, Morocco, Spain and Syria have been determined previously and we now report the first five genome sequences of FBNYV and associated alphasatellites from faba bean sampled in Tunisia...
November 17, 2017: Archives of Virology
https://www.readbyqxmd.com/read/29147487/a-study-on-the-immune-response-induced-by-a-dna-vaccine-encoding-mtb32c-hbha-antigen-of-mycobacterium-tuberculosis
#9
Roghayeh Teimourpour, Hadi Peeridogaheh, Amir Teimourpour, Mohsen Arzanlou, Zahra Meshkat
Objectives: Tuberculosis (TB) has still remained a global health issue. One third of the world's population is infected with tuberculosis and the current BCG vaccine has low efficiency; hence, it is necessary to develop a new vaccine against TB. The aim of the current study was to evaluate the efficiency of a novel DNA vaccine encoding Mtb32C-HBHA antigen in inducing specific immune responses against Mycobacterium tuberculosis. Materials and Methods: A DNA plasmid vaccine expressing Mtb32C-HBHA fusion protein was constructed and its ability in protein expression was examined by RT-PCR and Western blot methods...
October 2017: Iranian Journal of Basic Medical Sciences
https://www.readbyqxmd.com/read/29146938/computational-investigation-of-homologous-recombination-dna-repair-deficiency-in-sporadic-breast-cancer
#10
Yue Wang, Matthew H Ung, Sharon Cantor, Chao Cheng
BRCAness has important implications in the management and treatment of patients with breast and ovarian cancer. In this study, we propose a computational framework to measure the BRCAness of breast and ovarian tumor samples based on their gene expression profiles. We define a characteristic profile for BRCAness by comparing gene expression differences between BRCA1/2 mutant familial tumors and sporadic breast cancer tumors while adjusting for relevant clinical factors. With this BRCAness profile, our framework calculates sample-specific BRCA scores, which indicates homologous recombination (HR)-mediated DNA repair pathway activity of samples...
November 16, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29146545/werner-syndrome-wrn-gene-variants-and-their-association-with-altered-function-and-age-associated-diseases
#11
REVIEW
Michel Lebel, Raymond J Monnat
Werner syndrome (WS) is a heritable autosomal recessive human disorder characterized by the premature onset of several age-associated pathologies including cancer. The protein defective in WS patients, WRN, is encoded by a member of the human RECQ gene family that contains both a DNA exonuclease and a helicase domain. WRN has been shown to participate in several DNA metabolic pathways including DNA replication, recombination and repair, as well as telomere maintenance and transcription modulation. Here we review base pair-level genetic variation that has been documented in WRN, with an emphasis on non-synonymous coding single nucleotide polymorphisms (SNPs) and their associations with anthropomorphic features, longevity and disease risk...
November 13, 2017: Ageing Research Reviews
https://www.readbyqxmd.com/read/29146152/improved-antifungal-activity-of-barley-derived-chitinase-i-gene-that-overexpress-a-32kda-recombinant-chitinase-in-escherichia-coli-host
#12
Nida Toufiq, Bushra Tabassum, Muhammad Umar Bhatti, Anwar Khan, Muhammad Tariq, Naila Shahid, Idrees Ahmad Nasir, Tayyab Husnain
Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935bp full-length chitinase I gene...
October 31, 2017: Brazilian Journal of Microbiology: [publication of the Brazilian Society for Microbiology]
https://www.readbyqxmd.com/read/29145865/the-endonuclease-eepd1-mediates-synthetic-lethality-in-rad52-depleted-brca1-mutant-breast-cancer-cells
#13
Robert Hromas, Hyun-Suk Kim, Gurjit Sidhu, Elizabeth Williamson, Aruna Jaiswal, Taylor A Totterdale, Jocelyn Nole, Suk-Hee Lee, Jac A Nickoloff, Kimi Y Kong
BACKGROUND: Proper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5' endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52)...
November 16, 2017: Breast Cancer Research: BCR
https://www.readbyqxmd.com/read/29145860/a-new-single-step-protocol-for-rapid-baculovirus-driven-protein-production-in-insect-cells
#14
Judith Scholz, Sabine Suppmann
BACKGROUND: In the last three decades, the Baculovirus expression vector system (BEV) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression including approved vaccines and therapies. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. In this study we show that the conventional method to generate recombinant Baculovirus using a Tn7 transposition based system can be shortened to a single-step transfection-only procedure without further amplification...
November 16, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/29145649/single-molecule-compaction-of-megabase-long-chromatin-molecules-by-multivalent-cations
#15
Anatoly Zinchenko, Nikolay V Berezhnoy, Sai Wang, William M Rosencrans, Nikolay Korolev, Johan R C van der Maarel, Lars Nordenskiöld
To gain insight into the conformational properties and compaction of megabase-long chromatin molecules, we reconstituted chromatin from T4 phage DNA (165 kb) and recombinant human histone octamers (HO). The unimolecular compaction, induced by divalent Mg2+ or tetravalent spermine4+ cations, studied by single-molecule fluorescence microscopy (FM) and dynamic light scattering (DLS) techniques, resulted in the formation of 250-400 nm chromatin condensates. The compaction on this scale of DNA size is comparable to that of chromatin topologically associated domains (TAD) in vivo...
November 14, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29145618/the-histone-variant-h2a-z-promotes-initiation-of-meiotic-recombination-in-fission-yeast
#16
Shintaro Yamada, Kazuto Kugou, Da-Qiao Ding, Yurika Fujita, Yasushi Hiraoka, Hiroshi Murakami, Kunihiro Ohta, Takatomi Yamada
Meiotic recombination is initiated by programmed formation of DNA double strand breaks (DSBs), which are mainly formed at recombination hotspots. Meiotic DSBs require multiple proteins including the conserved protein Spo11 and its cofactors, and are influenced by chromatin structure. For example, local chromatin around hotspots directly impacts DSB formation. Moreover, DSB is proposed to occur in a higher-order chromatin architecture termed 'axis-loop', in which many loops protrude from cohesin-enriched axis...
November 14, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29145601/identification-and-functional-analysis-of-potential-prophage-derived-recombinases-for-genome-editing-in-lactobacillus-casei
#17
Yongping Xin, Tingting Guo, Yingli Mu, Jian Kong
Numerous LAB bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by replacement of a 167 bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5'-3' exonuclease (Redα/RecE), respectively...
November 14, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/29144403/the-non-homologous-end-joining-protein-paxx-acts-to-restrict-hsv-1-infection
#18
Ben J Trigg, Katharina B Lauer, Paula Fernandes Dos Santos, Heather Coleman, Gabriel Balmus, Daniel S Mansur, Brian J Ferguson
Herpes simplex virus 1 (HSV-1) has extensive interactions with the host DNA damage response (DDR) machinery that can be either detrimental or beneficial to the virus. Proteins in the homologous recombination pathway are known to be required for efficient replication of the viral genome, while different members of the classical non-homologous end-joining (c-NHEJ) pathway have opposing effects on HSV-1 infection. Here, we have investigated the role of the recently-discovered c-NHEJ component, PAXX (Paralogue of XRCC4 and XLF), which we found to be excluded from the nucleus during HSV-1 infection...
November 16, 2017: Viruses
https://www.readbyqxmd.com/read/29141206/role-of-the-pif1-pcna-complex-in-pol-%C3%AE-dependent-strand-displacement-dna-synthesis-and-break-induced-replication
#19
Olga Buzovetsky, Youngho Kwon, Nhung Tuyet Pham, Claire Kim, Grzegorz Ira, Patrick Sung, Yong Xiong
The S. cerevisiae Pif1 helicase functions with DNA polymerase (Pol) δ in DNA synthesis during break-induced replication (BIR), a conserved pathway responsible for replication fork repair and telomere recombination. Pif1 interacts with the DNA polymerase processivity clamp PCNA, but the functional significance of the Pif1-PCNA complex remains to be elucidated. Here, we solve the crystal structure of PCNA in complex with a non-canonical PCNA-interacting motif in Pif1. The structure guides the construction of a Pif1 mutant that is deficient in PCNA interaction...
November 14, 2017: Cell Reports
https://www.readbyqxmd.com/read/29138572/current-status-of-poly-adp-ribose-polymerase-inhibitors-and-future-directions
#20
REVIEW
Akihiro Ohmoto, Shinichi Yachida
Inhibitors of poly(ADP-ribose) polymerases (PARPs), which play a key role in DNA damage/repair pathways, have been developed as antitumor agents based on the concept of synthetic lethality. Synthetic lethality is the idea that cell death would be efficiently induced by simultaneous loss of function of plural key molecules, for example, by exposing tumor cells with inactivating gene mutation of BRCA-mediated DNA repair to chemically induced inhibition of PARPs. Indeed, three PARP inhibitors, olaparib, rucaparib and niraparib have already been approved in the US or Europe, mainly for the treatment of BRCA-mutant ovarian cancer...
2017: OncoTargets and Therapy
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