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Molecular cloning

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https://www.readbyqxmd.com/read/29151158/guidelines-to-reach-high-quality-purified-recombinant-proteins
#1
REVIEW
Carla Oliveira, Lucília Domingues
The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process...
November 18, 2017: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29149898/detection-of-plasmodium-falciparum-male-and-female-gametocytes-and-determination-of-parasite-sex-ratio-in-human-endemic-populations-by-novel-cheap-and-robust-rtqpcr-assays
#2
Federica Santolamazza, Pamela Avellino, Giulia Siciliano, Franck Adama Yao, Fabrizio Lombardo, Jean Bosco Ouédraogo, David Modiano, Pietro Alano, Valentina Dianora Mangano
BACKGROUND: The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. AIM AND OBJECTIVES: The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts...
November 17, 2017: Malaria Journal
https://www.readbyqxmd.com/read/29149486/discovery-of-vg-and-vgr-gene-expression-profiles-related-to-host-plant-suitability-of-spodoptera-exigua
#3
Jing Zhao, Yang Sun, Liubin Xiao, Yongan Tan, Yiping Jiang, Lixin Bai
BACKGROUND: Spodoptera exigua, a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and host plant is a basic and important component of early forecasting of beet armyworm. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. RESULTS: In this study, the key parameters of S. exigua grown on distinct hosts were investigated; host plants could be ranked as lettuce >shallot > tomato > celery in their order of suitability...
November 17, 2017: Pest Management Science
https://www.readbyqxmd.com/read/29145427/do-microplastic-particles-affect-daphnia-magna-at-the-morphological-life-history-and-molecular-level
#4
Hannes K Imhof, Jakub Rusek, Michaela Thiel, Justyna Wolinska, Christian Laforsch
Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of ~40 μm were supplied at a low concentration (1% of the food particles) leading to an average of ~30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation...
2017: PloS One
https://www.readbyqxmd.com/read/29143132/thermostable-and-highly-specific-l-aspartate-oxidase-from-thermococcus-litoralis-dsm-5473-cloning-overexpression-and-enzymological-properties
#5
Tsubasa Washio, Tadao Oikawa
We successfully expressed the L-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed L-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an L-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria...
November 15, 2017: Extremophiles: Life Under Extreme Conditions
https://www.readbyqxmd.com/read/29142737/cloning-expression-and-identification-of-ktx-sp4-a-selective-kv1-3-peptidic-blocker-from-scorpiops-pococki
#6
Yan Zou, Feng Zhang, Yaxian Li, Yuanfang Wang, Yi Li, Zhengtao Long, Shujuan Shi, Li Shuai, Jiukai Liu, Zhiyong Di, Shijin Yin
Background: Specific and selective peptidic blockers of Kv1.3 channels can serve as a valuable drug lead for treating T cell-mediated autoimmune diseases, and scorpion venom is an important source of kv1.3 channel inhibitors. Through conducting transcriptomic sequencing for the venom gland of Scorpiops pococki from Xizang province of China, this research aims to discover a novel functional gene encoding peptidic blocker of Kv1.3, and identify its function. Results: We screened out a new peptide toxin KTX-Sp4 which had 43 amino acids including six cysteine residues...
2017: Cell & Bioscience
https://www.readbyqxmd.com/read/29142124/truncating-the-gp41-cytoplasmic-tail-of-siv-decreases-sensitivity-to-neutralizing-antibodies-without-increasing-envelope-content-of-virions
#7
Ellen White, Fan Wu, Elena Chertova, Julian Bess, James D Roser, Jeffrey D Lifson, Vanessa M Hirsch
An incomplete understanding of native HIV and SIV envelope glycoprotein (Env) impedes the development of structural models of Env and vaccine design. This shortcoming is due in part to the low number of Env trimers on virus particles. For SIV, this low expression can be counteracted by truncating the cytoplasmic tail (CT) of Env. CT truncation has been shown to increase Env incorporation into the virion and is commonly used in vaccine and imaging studies, but its effects on viral antigenicity have not been fully elucidated...
November 15, 2017: Journal of Virology
https://www.readbyqxmd.com/read/29142081/genetic-and-molecular-regulation-of-extrasynaptic-gaba-a-receptors-in-the-brain-therapeutic-insights-for-epilepsy
#8
Shu-Hui Chuang, Doodipala Samba Reddy
GABA-A receptors play a pivotal role in many brain diseases. Epilepsy is caused by acquired conditions and genetic defects in GABA receptor channels regulating neuronal excitability in the brain. The latter is referred to as GABA channelopathies. In the last two decades, major advances have been made in the genetics of epilepsy. The presence of specific GABAergic genetic abnormalities leading to some of the classical epileptic syndromes has been identified. Advances in molecular cloning and recombinant systems have helped characterize mutations in GABA-A receptor subunit genes in clinical neurology...
November 15, 2017: Journal of Pharmacology and Experimental Therapeutics
https://www.readbyqxmd.com/read/29141193/evolution-of-the-clock-and-bmal1-genes-in-a-subterranean-rodent-species-lasiopodomys-mandarinus
#9
Hong Sun, Yifeng Zhang, Yuhua Shi, Yangwei Li, Wei Li, Zhenlong Wang
Lasiopodomys mandarinus, a subterranean rodent, spends its entire life underground. To test whether the CLOCK and BMAL1 genes of L. mandarinus have undergone adaptive evolution to underground darkness, we cloned and analyzed their complete cDNA sequences, using Lasiopodomys brandtii as a control. The phylogenetic trees of the CLOCK and BMAL1 genes were similar to the trees of the conserved Cyt b gene,further, L. mandarinus clustered with L. brandtii and Microtus ochrogaster in the phylogenetic tree. The Q-rich region of the CLOCK gene in L...
November 12, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29141051/molecular-and-epidemiological-characterization-of-carbapenemase-producing-enterobacteriaceae-in-norway-2007-to-2014
#10
Ørjan Samuelsen, Søren Overballe-Petersen, Jørgen Vildershøj Bjørnholt, Sylvain Brisse, Michel Doumith, Neil Woodford, Katie L Hopkins, Bettina Aasnæs, Bjørg Haldorsen, Arnfinn Sundsfjord
The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. Here we present associated patient data and molecular, epidemiological and phenotypic characteristics of all CPE isolates in Norway from 2007 to 2014 confirmed at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance. All confirmed CPE isolates were characterized pheno- and genotypically, including by whole genome sequencing (WGS). Patient data were reviewed retrospectively. In total 59 CPE isolates were identified from 53 patients...
2017: PloS One
https://www.readbyqxmd.com/read/29140998/using-whole-genome-sequencing-to-study-american-foulbrood-epidemiology-in-honeybees
#11
Joakim Ågren, Marc Oliver Schäfer, Eva Forsgren
American foulbrood (AFB), caused by Paenibacillus larvae, is a devastating disease in honeybees. In most countries, the disease is controlled through compulsory burning of symptomatic colonies causing major economic losses in apiculture. The pathogen is endemic to honeybees world-wide and is readily transmitted via the movement of hive equipment or bees. Molecular epidemiology of AFB currently largely relies on placing isolates in one of four ERIC-genotypes. However, a more powerful alternative is multi-locus sequence typing (MLST) using whole-genome sequencing (WGS), which allows for high-resolution studies of disease outbreaks...
2017: PloS One
https://www.readbyqxmd.com/read/29140111/purging-exhausted-virus-specific-cd8-t-cell-phenotypes-by-somatic-cell-reprogramming
#12
Joshua Chan, Patrick Y Kim, Emiko Kranz, Yoshiko Nagaoka, YooJin Lee, Jing Wen, Heidi J Elsaesser, Meng Qin, David G Brooks, Gene-Errol Ringpis, Irvin S Y Chen, Masakazu Kamata
Cytotoxic T cells are critical in controlling virus infections. However, continuous antigen stimulation and negative regulatory factors cause CD8 T cells to enter a dysfunctional state (T cell exhaustion), resulting in viral persistence. We hypothesized that the exhausted T cell state could be molecularly rejuvenated using a somatic cell reprogramming technology, which is technically able to convert any types of cells to induced pluripotent stem cells (iPSCs), to regenerate functional T cells capable of purging chronic infection...
November 2017: AIDS Research and Human Retroviruses
https://www.readbyqxmd.com/read/29139543/molecular-cloning-and-expression-analysis-of-sox3-during-gonad-and-embryonic-development-in-misgurnus-anguillicaudatus
#13
Xiaohua Xia, Weiran Huo, Ruyan Wan, Linxia Zhang, Xiaopei Xia, Zhongjie Chang
Sox3 is a single-exon gene located on the X chromosome in most vertebrates. It belongs to the SoxB1 subfamily, which is part of the larger Sox family. Previous studies have revealed that Sox3 is expressed in many fish species. However, how Sox3 influences the development of Misgurnus anguillicaudatus remains unknown. In this study, a Sox3 homologue, termed MaSox3, was cloned from the brain of M. anguillicaudatus using homology-based cloning and the rapid amplification of cDNA ends method. Sequence analysis reveals that MaSox3 encodes a hydrophilic protein, which contains a characteristic HMG-box DNA-binding domain of 79 amino acids, and shares high homology with Sox3 in other species...
2017: International Journal of Developmental Biology
https://www.readbyqxmd.com/read/29139018/vaccination-against-hydatidosis-molecular-cloning-and-optimal-expression-of-the-eg95nc-recombinant-antigen-in-escherichia-coli
#14
M Jazouli, M Lightowlers, C G Gauci, K Tadlaoui, A Belmlih, M M Ennaji, M Elharrak
Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries...
November 14, 2017: Protein Journal
https://www.readbyqxmd.com/read/29138998/crafting-socialist-embryology-dialectics-aquaculture-and-the-diverging-discipline-in-maoist-china-1950-1965
#15
Lijing Jiang
In the 1950s, embryology in socialist China underwent a series of changes that adjusted the disciplinary apparatus to suit socialism and the national goal of self-reliance. As the Communist state called on scientists to learn from the Soviets, embryologists' comprehensive view on heredity, which did not contradict Trofim Lysenko (1898-1976)'s doctrines, provided a space for them to advance their discipline. Leading scientists, often trained abroad in the tradition of experimental embryology, rode on the tides of Maoist ideology and repositioned their research...
November 7, 2017: History and Philosophy of the Life Sciences
https://www.readbyqxmd.com/read/29138983/cloning-expression-analysis-and-enzyme-activity-assays-of-the-%C3%AE-carbonic-anhydrase-gene-from-chlamydomonas-sp-ice-l
#16
Changfeng Qu, Yingying He, Zhou Zheng, Meiling An, Lulu Li, Xixi Wang, Xiaodong He, Yibin Wang, Fangming Liu, Jinlai Miao
The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0...
November 14, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/29137281/hic-5-regulates-epithelial-to-mesenchymal-transition-in-ovarian-cancer-cells-in-a-tgf%C3%AE-1-independent-manner
#17
Razan Sheta, Zhi-Qiang Wang, Magdalena Bachvarova, Marie Plante, Jean Gregoire, Marie-Claude Renaud, Alexandra Sebastianelli, Stephane Gobeil, Chantale Morin, Elizabeth Macdonald, Barbara Vanderhyden, Dimcho Bachvarov
The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. We have previously identified the hydrogen peroxide-inducible clone-5 (Hic-5) gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. Hic-5 is a focal adhesion scaffold protein and has been primarily studied for its role as a key mediator of TGF-β-induced epithelial-to-mesenchymal transition (EMT) in epithelial cells of both normal and malignant origin; however, its role in EOC has been never investigated...
October 10, 2017: Oncotarget
https://www.readbyqxmd.com/read/29137147/development-of-pgemini-a-plant-gateway-destination-vector-allowing-the-simultaneous-integration-of-two-cdna-via-a-single-lr-clonase-reaction
#18
Marino Exposito-Rodriguez, Philippe P Laissue, Patricia E López-Calcagno, Philip M Mullineaux, Christine A Raines, Andrew J Simkin
Gateway technology has been used to facilitate the generation of a large number of constructs for the modification of plants for research purposes. However, many of the currently available vectors only allow the integration of a single cDNA of interest into an expression clone. The ability to over-express multiple genes in combination is essential for the study of plant development where several transcripts have a role to play in one or more metabolic processes. The tools to carry out such studies are limited, and in many cases rely on the incorporation of cDNA into expression systems via conventional cloning, which can be both time consuming and laborious...
November 12, 2017: Plants (Basel, Switzerland)
https://www.readbyqxmd.com/read/29136398/molecular-cloning-and-epigenetic-change-detection-of-kiss1-during-seasonal-reproduction-in-chinese-indigenous-sheep
#19
Xiaoyun He, Qiuyue Liu, Xiaoyu Li, Xiaofei Guo, Xiangyu Wang, Wenping Hu, Ran Di, Mingxing Chu
Like most seasonal domesticated species, sheep are short-day breeders, which means that the reproduction axis is activated by short days. The annual photoperiodic cycle affects the amount of daylength information that is transmitted to the hypothalamic-pituitary-gonadal (HPG) axis by regulating pulsatile secretion of gonadotrophin-releasing hormone from the hypothalamus. Kisspeptin, which is encoded by Kiss1, plays a major role in reproductive seasonality. Based on results from our previous Solexa sequencing data obtained from Tan (T) and Small Tail Han (STH) sheep during anoestrus and the breeding season, full-length mRNA information for ovine Kiss1 was obtained; 894bp in T sheep and 1145bp in STH sheep...
November 15, 2017: Reproduction, Fertility, and Development
https://www.readbyqxmd.com/read/29134647/serial-profiling-of-circulating-tumor-dna-for-optimization-of-anti-vegf-chemotherapy-in-metastatic-colorectal-cancer-patients
#20
Masami Yamauchi, Yuji Urabe, Atsushi Ono, Daiki Miki, Hidenori Ochi, Kazuaki Chayama
Understanding the molecular changes in tumors in response to anti-VEGF chemotherapy is crucial for optimization of the treatment strategy for metastatic colorectal cancer. We prospectively investigated changes in the amount and constitution of circulating tumor DNA (ctDNA) in serial peripheral blood samples during chemotherapy. Sixty-one plasma samples taken at different time points (baseline, remission, and post-progression) and pre-treatment tumor samples were collected from 21 patients who received bevacizumab-containing first-line chemotherapy...
November 14, 2017: International Journal of Cancer. Journal International du Cancer
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