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Molecular cloning

Fufa Qu, Zhiming Xiang, Yang Zhang, Jun Li, Shu Xiao, Yuehuan Zhang, Fan Mao, Haitao Ma, Ziniu Yu
p38 mitogen-activated protein kinases (MAPKs) are conserved serine/threonine-specific kinases that are activated by various extracellular stimuli and play crucial regulatory roles in immunity, development and homeostasis. However, the function of p38s in mollusks, the second most diverse group of animals, is still poorly understood. In this study, a novel molluscan p38 (designated Chp38) was cloned and characterized from the Hong Kong oyster Crassostrea hongkongensis. Its full-length cDNA encoded a putative protein of 353 amino acids with a calculated molecular weight of approximately 40...
October 18, 2016: Molecular Immunology
Rui Seixas, Tânia Raquel Santos, Jorge Machado, Luís Tavares, Fernando Bernardo, Teresa Semedo-Lemsaddek, Manuela Oliveira
The increase in prevalence of Salmonella 1,4,[5],12:i:- related infections over the last few years has been considered a public health issue in many European countries, especially as this serovar may be associated with tetraresistance to ampicillin, streptomycin, sulfonamides, and tetracyclines (R-type ASSuT). Salmonella 1,4,[5],12:i:- isolates (n = 187) obtained by the Portuguese National Laboratory from different sources, including human clinical cases (n = 170), veterinary (n = 10), environmental (n = 6), and food samples (n = 1), were collected from 15 districts between 2006 and 2011...
October 21, 2016: Foodborne Pathogens and Disease
Juliane Mundorf, Mirka Uhlirova
Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique...
October 6, 2016: Journal of Visualized Experiments: JoVE
Wasay M Shaikh Qureshi, Lianjie Miao, David Shieh, Jingjing Li, Yangyang Lu, Saiyang Hu, Margarida Barroso, Joseph Mazurkiewicz, Mingfu Wu
Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution...
October 7, 2016: Journal of Visualized Experiments: JoVE
Ewa Szymańska, Paulina Chałupnik, Katarzyna Szczepańska, Ana Maria Cuñado Moral, Darryl S Pickering, Birgitte Nielsen, Tommy N Johansen, Katarzyna Kieć-Kononowicz
A new series of carboxyaryl-substituted phenylalanines was designed, synthesized and pharmacologically characterized in vitro at native rat ionotropic glutamate receptors as well as at cloned homomeric kainate receptors GluK1-GluK3. Among them, six compounds bound to GluK1 receptor subtypes with reasonable affinity (Ki values in the range of 4.9-7.5μM). A structure-activity relationship (SAR) for the obtained series, focused mainly on the pharmacological effect of structural modifications in the 4- and 5-position of the phenylalanine ring, was established...
September 30, 2016: Bioorganic & Medicinal Chemistry Letters
Y J Mu, B F Zhang, J Y Zhao, Z Q Wang, S L Xia, X Y Huang, B L Xu
Objective: To understand the distribution of Salmonella in external environment in Henan province, and explore the distribution of different serotypes of the Salmonella and their homology. Methods: A total of 4 488 samples were collected form animal dung, meat products and kitchen utensils, and identified by biochemical tests and serotyped by serum agglutination reaction. The predominant serotypes were further typed by PFGE. Results: A total of 324 Salmonella strains were detected in these samples, the detection rate was 7...
October 10, 2016: Zhonghua Liu Xing Bing Xue za Zhi, Zhonghua Liuxingbingxue Zazhi
Ahmed Abdel Megeed, M Ali Hayssam, Mohamed Z M Salem, Mohamed S El-Shikh, Ibrahim Ahmed Talea, Yahya A Alogaibi
Multidrug-resistant (MDR) Acinetobacter baumannii infections are a great public health concern and demand continuous surveillance and antibiotic stewardship. Virulence traits and the pathogenicity of Acinetobacter are less studied compared with the molecular epidemiological and antibiotic resistance profile of this organism. In our present study, we investigated the primary characteristics contributing to the virulence of MDR A. baumannii isolates and compared them with avirulent isolates. A total of 32 well-characterized MDR A...
October 19, 2016: Journal of AOAC International
Lasse Kjær, Sabrina Cordua, Morten O Holmström, Mads Thomassen, Torben A Kruse, Niels Pallisgaard, Thomas S Larsen, Karin de Stricker, Vibe Skov, Hans C Hasselbalch
Discovery of somatic mutations in the calreticulin gene (CALR) has identified a subgroup of Philadelphia-negative chronic myeloproliferative neoplasms (MPN) with separate haematological characteristics and prognosis. CALR mutations serve as novel markers both of diagnostic value and as targets for monitoring molecular responses during therapy. Interferon-α (IFN) selectively targets the malignant clone in a subset of MPN patients and can induce both haematological and molecular remissions in CALR mutated essential thrombocythemia (ET) patients...
2016: PloS One
Shuangshuang Zhao, Lei Zhao, Fengxia Liu, Yongzhen Wu, Zuofeng Zhu, Chuanqing Sun, Lubin Tan
Grain yield in rice (Oryza sativa L.) is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should to be addressed. Here we identified two allelic rice mutants with narrow and semi-rolled leaves, named narrow and rolled leaf 2-1 (nrl2-1) and nrl2-2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration and microspore development, and the formation of more slender seeds compared with the wild type (WT)...
October 20, 2016: Journal of Integrative Plant Biology
Lakshmi Kasirajan, Kalaivaani Aruchamy, Prathima P Thirugnanasambandam, Selvi Athiappan
Sugarcane (Saccharum spp.) is one of the highest biomass-producing plant and the best lignocellulosic feedstock for ethanol production. To achieve more efficient conversion of biomass to ethanol, a better understanding of the main factors affecting biomass recalcitrance is needed. Therefore, with this objective, here, we report a systematic study on lignin content, deposition, identification, and cloning of genes involved in lignin biosynthesis and their differential expression in five sugarcane clones, EC11003, EC11010, IK 76-91, IK 76-99, and Co 86032...
October 19, 2016: Applied Biochemistry and Biotechnology
Joseph F O'Grady, Laura S Hoelters, Martin T Swain, David C Wilcockson
BACKGROUND: Talitrus saltator is an amphipod crustacean that inhabits the supralittoral zone on sandy beaches in the Northeast Atlantic and Mediterranean. T. saltator exhibits endogenous locomotor activity rhythms and time-compensated sun and moon orientation, both of which necessitate at least one chronometric mechanism. Whilst their behaviour is well studied, currently there are no descriptions of the underlying molecular components of a biological clock in this animal, and very few in other crustacean species...
2016: PeerJ
Saroor A Patel, Sakari Vanharanta
Genetic analyses of cancer progression in patient samples and model systems have thus far failed to identify specific mutational drivers of metastasis. Yet, at least in experimental systems, metastatic cancer clones display stable traits that can facilitate progression through the many steps of metastasis. How cancer cells establish and maintain the transcriptional programmes required for metastasis remains mostly unknown. Emerging evidence suggests that metastatic traits may arise from epigenetically altered transcriptional output of the oncogenic signals that drive tumour initiation and early progression...
October 8, 2016: Molecular Oncology
Partha Pratim Bose, Prakash Kumar
In high-throughput biotechnology and structural biology, molecular cloning is an essential prerequisite for attaining high yields of recombinant protein. However, a rapid, cost-effective, easy clone screening protocol is still required to identify colonies with desired insert along with a cross check method to certify the expression of the desired protein as the end product. We report an easy, fast, sensitive and cheap visual clone screening and protein expression cross check protocol employing gold nanoparticle based plasmonic detection phenomenon...
October 15, 2016: Analytical Biochemistry
Cameron S Gordon, Nandhakishore Rajagopalan, Eddy P Risseeuw, Marci Surpin, Fraser J Ball, Carla J Barber, Leann M Buhrow, Shawn M Clark, Jonathan E Page, Chris D Todd, Suzanne R Abrams, Michele C Loewen
Abscisic acid (ABA) is a well-characterized plant hormone, known to mediate developmental aspects as well as both abiotic and biotic stress responses. Notably, the exogenous application of ABA has recently been shown to increase susceptibility to the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight (FHB) in wheat and other cereals. However roles and mechanisms associated with ABA's modulation of pathogen responses remain enigmatic. Here the identification of putative ABA receptors from available genomic databases for Triticum aestivum (bread wheat) and Brachypodium distachyon (a model cereal) are reported...
2016: PloS One
Gabriela Barcenas-Morales, Peter Jandus, Rainer Döffinger
PURPOSE OF REVIEW: Concise overview of the field of anticytokine autoantibodies with a focus on recent developments. RECENT FINDINGS: Advances in particular in the analysis of autoantibodies to IFNγ, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-1 are presented. The target epitope for anti-IFNγ autoantibodies has been found to have high homology to a protein from Aspergillus suggesting molecular mimicry as a mechanism of breaking self-tolerance...
October 13, 2016: Current Opinion in Allergy and Clinical Immunology
Jinhua Sun, Yinglin Chen, Feiyue Qin, Xueting Guan, Wei Xu, Liangmei Xu
Interferons have attracted considerable attention due to their vital roles in the host immune response and low induction of antibiotic resistance. In this study, total RNA was extracted from spleen cells of chicken embryos inoculated with Newcastle disease vaccine, and the full-length chicken interferon-γ (ChIFN-γ) gene was amplified by RT-PCR. The full complementary DNA sequence of the ChIFN-γ gene was 495 bp long and was cloned into the prokaryotic expression vector pProEX™HTb . The plasmid was transformed into Escherichia coli DH5α and the expression of ChIFN-γ was induced by isopropyl β-D-1-thiogalactopyranoside...
October 17, 2016: Animal Science Journal, Nihon Chikusan Gakkaihō
Ji-Hua Yu, Yang-Yang Li, Mian Xiang, Jian-Quan Zhu, Xin-He Huang, Wan-Jun Wang, Rui Tan, Jia-Yu Zhou, Hai Liao
OBJECTIVES: To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine. RESULTS: The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin...
October 17, 2016: Biotechnology Letters
Maelin da Silva, Patricia Barbosa, Roberto F Artoni, Eliana Feldberg
Gymnotidae is a family of electric fish endemic to the Neotropics consisting of 2 genera: Electrophorus and Gymnotus. The genus Gymnotus is widely distributed and is found in all of the major Brazilian river systems. Physical and molecular mapping data for the ribosomal DNA (rDNA) in this genus are still scarce, with its chromosomal location known in only 11 species. As other species of Gymnotus with 2n = 54 chromosomes from the Paraná-Paraguay basin, G. mamiraua was found to have a large number of 5S rDNA sites...
October 18, 2016: Cytogenetic and Genome Research
Yin-Ku Liang, Liu-Jiao Bian
Kringle 5, the fifth fragment of plasminogen, is known to be important for inhibiting the proliferation and migration of vascular endothelial cell (VEC), while not having any effects on normal endothelial cells. Therefore, it may be a potential tumor therapy candidate. However, the ligand of the Kringle 5 in VEC has not yet been identified. In this study, the possible ligand of Kringle 5 in vitro was screened and validated using Ph.D.-7 phage display peptide library with molecular docking, along with surface plasma resonance (SPR)...
2016: PloS One
Arvind Sivasubramanian, Patricia Estep, Heather Lynaugh, Yao Yu, Adam Miles, Josh Eckman, Kevin Schutz, Crystal Piffath, Nadthakarn Boland, Rebecca Hurley Niles, Stéphanie Durand, Todd Boland, Maximiliano Vásquez, Yingda Xu, Yasmina Abdiche
Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad "epitope coverage" increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or "bins" and prioritize leads for further characterization and optimization...
October 17, 2016: MAbs
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