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Molecular cloning

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https://www.readbyqxmd.com/read/29331678/microrna-320-enhances-radiosensitivity-of-glioma-through-down-regulation-of-sirtuin-type-1-by-directly-targeting-forkhead-box-protein-m1
#1
Tengfei Li, Ji Ma, Xinwei Han, Yongxu Jia, Huifeng Yuan, Shaofeng Shui, Dong Guo
Glioma is the most common cancer in human brain system and seriously threatens human health. miRNA-320 has been demonstrated to be closely correlated with the development of glioma. However, its effect and molecular mechanism underlying radioresistance have not been fully elucidated in glioma. Here, RT-qPCR assay was used to assess the expressions of miR-320 and forkhead box protein M1 (FoxM1) mRNA in glioma tumor tissues and cells. The effects of miR-320, FoxM1 and sirtuin type 1 (Sirt1) on radiosensitivity in glioma cells were evaluated by clone formation assay, apoptosis assay, histone H2AX phosphorylation level (γH2AX) detection and caspase 3 activity analysis, respectively...
January 10, 2018: Translational Oncology
https://www.readbyqxmd.com/read/29331528/expression-of-a-thermotolerant-laccase-from-pycnoporus-sanguineus-in-trichoderma-reesei-and-its-application-in-the-degradation-of-bisphenol-a
#2
Jie Zhao, Shenquan Zeng, Ying Xia, Liming Xia
The laccase gene from Pycnoporus sanguineus was cloned and inserted between the strong Pcbh1 promoter and the Tcbh1 terminator from Trichoderma reesei to form the recombinant plasmid pCH-lac. Using Agrobacterium-mediated technique, the pCH-lac was integrated into the chromosomes of T. reesei. Twenty positive transformants were obtained by employing hygromycin B as a selective agent. PCR was used to confirm that the laccase gene was integrated into the chromosomal DNA of T. reesei. Laccase production by recombinant transformants was performed in shaking flasks, and the activity of laccase reached 8...
January 10, 2018: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/29331367/molecular-characterization-and-differential-expression-analysis-of-interleukin-1%C3%AE-from-ovis-aries
#3
Yi-Ming Shui, Shi-Ying Lu, Xing Guo, Xi-Lin Liu, Bao-Quan Fu, Pan Hu, Lin-Lin Qu, Nan-Nan Liu, Yan-Song Li, Lu-Lu Wang, Fei-Fei Zhai, Dan-Di Ju, Zeng-Shan Liu, Yu Zhou, Hong-Lin Ren
The interleukin-1 family is an important component of the innate immune system and plays an important role in regulating immune responses on the invasion of intracellular parasites in the acquired immune system. Interleukin 1β (IL-1β) is one of the members of the IL-1 family that predominantly activates downstream signaling pathways to play immunological functions of stimulating T and B lymphocyte activation and promoting the various syntheses of inflammatory substances in conjunction with other cytokines...
January 10, 2018: Microbial Pathogenesis
https://www.readbyqxmd.com/read/29331077/optimized-paired-sgrna-cas9-cloning-and-expression-cassette-triggers-high-efficiency-multiplex-genome-editing-in-kiwifruit
#4
Zupeng Wang, Shuaibin Wang, Dawei Li, Qiong Zhang, Li Li, Caihong Zhong, Yifei Liu, Hongwen Huang
Kiwifruit is an important fruit crop; however, technologies for its functional genomic and molecular improvement are limited. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability is variable depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Optimizing conditions for its use within a particular species is therefore needed to achieve highly efficient genome editing...
January 13, 2018: Plant Biotechnology Journal
https://www.readbyqxmd.com/read/29331014/biochemical-characterization-of-recombinant-thermostable-cohnella-sp-a01-%C3%AE-glucanase
#5
Meysam Rezaie, Saeed Aminzadeh, Farid Heidari, Masoud Mashhadi Akbar Boojar, Ali Asghar Karkhane
Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are release by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01. Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3)...
January 13, 2018: Iranian Biomedical Journal
https://www.readbyqxmd.com/read/29329981/molecular-cloning-and-characterization-of-leucine-aminopeptidase-gene-from-taenia-pisiformis
#6
Shaohua Zhang, Xuepeng Cai, Xuenong Luo, Shuai Wang, Aijiang Guo, Junling Hou, Run Wu
Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa...
January 9, 2018: Experimental Parasitology
https://www.readbyqxmd.com/read/29326121/modeling-multiple-myeloma-bone-marrow-interactions-and-response-to-drugs-in-a-3d-surrogate-microenvironment
#7
Daniela Belloni, Silvia Heltai, Maurilio Ponzoni, Antonello Villa, Barbara Vergani, Lorenza Pecciarini, Magda Marcatti, Stefania Girlanda, Giovanni Tonon, Fabio Ciceri, Federico Caligaris-Cappio, Marina Ferrarini, Elisabetta Ferrero
Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells...
January 11, 2018: Haematologica
https://www.readbyqxmd.com/read/29326013/a-cluster-of-multidrug-resistant-mycobacterium-tuberculosis-among-patients-arriving-in-europe-from-the-horn-of-africa-a-molecular-epidemiological-study
#8
Timothy M Walker, Matthias Merker, Astrid M Knoblauch, Peter Helbling, Otto D Schoch, Marieke J van der Werf, Katharina Kranzer, Lena Fiebig, Stefan Kröger, Walter Haas, Harald Hoffmann, Alexander Indra, Adrian Egli, Daniela M Cirillo, Jérôme Robert, Thomas R Rogers, Ramona Groenheit, Anne T Mengshoel, Vanessa Mathys, Marjo Haanperä, Dick van Soolingen, Stefan Niemann, Erik C Böttger, Peter M Keller
BACKGROUND: The risk of tuberculosis outbreaks among people fleeing hardship for refuge in Europe is heightened. We describe the cross-border European response to an outbreak of multidrug-resistant tuberculosis among patients from the Horn of Africa and Sudan. METHODS: On April 29 and May 30, 2016, the Swiss and German National Mycobacterial Reference Laboratories independently triggered an outbreak investigation after four patients were diagnosed with multidrug-resistant tuberculosis...
January 8, 2018: Lancet Infectious Diseases
https://www.readbyqxmd.com/read/29325077/integrating-restriction-site-associated-dna-sequencing-rad-seq-with-morphological-cladistic-analysis-clarifies-evolutionary-relationships-among-major-species-groups-of-bee-orchids
#9
Richard M Bateman, Gábor Sramkó, Ovidiu Paun
Background and Aims: Bee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus...
January 9, 2018: Annals of Botany
https://www.readbyqxmd.com/read/29323436/molecular-characterization-and-gene-silencing-of-laccase-1-in-the-grain-aphid-sitobion-avenae
#10
Yong Zhang, Jia Fan, Frédéric Francis, Julian Chen
Laccase 1 (Lac1), a polyphenol oxidase, has been proposed to be involved in insect iron metabolism and immunity responses. However, little information is available on the roles of Lac 1 in insect-plant interactions. The grain aphid Sitobion avenae is one of the most destructive pests of cereal, directly drawing phloem sap and transmitting viruses. In the present study, we first cloned the open reading frame (ORF) of Lac 1 from S. avenae, and the putative protein sequence was predicted to have a carboxyl-terminal transmembrane domain...
January 11, 2018: Archives of Insect Biochemistry and Physiology
https://www.readbyqxmd.com/read/29321316/two-residues-in-nsp9-contribute-to-the-enhanced-replication-and-pathogenicity-of-highly-pathogenic-porcine-reproductive-and-respiratory-syndrome-virus
#11
Kuan Zhao, Jia-Cong Gao, Jun-Yao Xiong, Jin-Chao Guo, Yong-Bo Yang, Cheng-Gang Jiang, Yan-Dong Tang, Zhi-Jun Tian, Xue-Hui Cai, Guang-Zhi Tong, Tong-Qing An
Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) possesses greater replicative capacity and pathogenicity than classical PRRSV. However, the factors that lead to enhanced replication and pathogenicity remain unclear. In our study, an alignment of all available full-length sequences of North-American type PRRSVs (n = 204) revealed two consistent amino acid mutations that differed between HP-PRRSV and classical PRRSV and were located at positions 519 and 544 in non-structural protein 9...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29321314/hla-class-i-downregulation-by-hiv-1-variants-from-subtype-c-transmission-pairs
#12
Zachary Ende, Martin J Deymier, Daniel T Claiborne, Jessica L Prince, Daniela C Mónaco, William Kilembe, Susan A Allen, Eric Hunter
HIV-1 downregulates HLA-A and HLA-B from the surface of infected cells primarily to evade CD8 T cell recognition. HLA-C was thought to remain on the cell surface and bind inhibitory killer immunoglobulin-like receptors, preventing NK cell-mediated suppression. However, a recent study found HIV-1 primary viruses have the capacity to downregulate HLA-C. The goal of this study was to assess the heterogeneity of HLA-A, HLA-B and HLA-C downregulation among full-length primary viruses from six chronically infected and six newly infected individuals from transmission pairs, and to determine whether transmitted/founder variants exhibit common HLA class I downregulation characteristics...
January 10, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29319227/fructokinase-like-protein-1-interacts-with-trxz-to-regulate-chloroplast-development-in-rice
#13
Lei He, Sen Zhang, Zhennan Qiu, Juan Zhao, Wendan Nie, Haiyan Lin, Zhengge Zhu, Dali Zeng, Qian Qian, Li Zhu
Chloroplast genes are transcribed by the PEP or NEP. FRUCTOKINASE-LIKE PROTEINS (FLNs) are phosphofructokinase-B (PfkB)-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLNs activity in rice remain elusive. Previously, we identified and characterized a heat-stress sensitive albino (hsa1) mutant in rice. Map-based cloning revealed that HSA1 encodes a putative OsFLN2. Here, we further demonstrated that knockdown or knockout of the OsFLN1, a close homolog of HSA1/OsFLN2, considerably inhibits chloroplast biogenesis and the fln1 knockout mutants created by CRISPR/Cas9 exhibit severe albino phenotype and seedling lethality...
January 10, 2018: Journal of Integrative Plant Biology
https://www.readbyqxmd.com/read/29319165/long-noncoding-rna-neat1-regulate-papillary-thyroid-cancer-progression-by-modulating-mir-129-5p-klk7-expression
#14
Hong Zhang, Yuechang Cai, Li Zheng, Zhanlei Zhang, Xiaofeng Lin, Ningyi Jiang
PURPOSE: Considering the dilemma in papillary thyroid cancer treatment, this study intended to find solution in molecular respect. By probing into lncRNA-NEAT1/miR-129-5p/ KLK7 interaction, this study would provide new targets for future treatment. METHODS: Microarray analysis and R language package were applied to select possible lncRNA and miRNA. Luciferase reporter assay and RNA pull-down test were employed in the detection of target relationship between lncRNA and miRNA...
January 10, 2018: Journal of Cellular Physiology
https://www.readbyqxmd.com/read/29318516/cloning-and-expression-of-h-influenzae-49247-iga-protease-in-e-coli
#15
Honglian Wang, Xia Zhong, Jianchun Li, Menglian Zhu, Lu Wang, Xingli Ji, Junming Fan, Li Wang
IgA protease is secreted by various mucosal pathogenic bacteria which can cleave human immunoglobulin A1 (IgA1) in its hinge region. In addition to be considered as a virulence factor, it's reported that IgA protease can also be used for IgA nephropathy (IgAN) treatment. Our previous study identified bacteria H. influenzae 49247 expressed high activity of IgA protease with promised application in IgAN therapy. In this study, we cloned the IgA protease gene of H. influenzae 49247 with degenerate primers. Alignment analysis indicated that H...
January 9, 2018: Molecular Biotechnology
https://www.readbyqxmd.com/read/29317874/molecular-cloning-expression-and-characterization-of-plasmid-encoding-rhomboid-4-rom4-of-tachyzoite-of-toxoplasma-gondii-rh-strain
#16
Mohammad Taghi Rahimi, Shahabeddin Sarvi, Mahdi Sharif, Saeid Abediankenari, Ehsan Ahmadpour, Reza Valadan, Ahmad Daryani
Background: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4) proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein. Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5' end of forward and reverse primers, respectively...
October 2017: Iranian Journal of Parasitology
https://www.readbyqxmd.com/read/29317320/molecular-cloning-characterization-and-expression-analysis-of-the-three-cysteine-and-glycine-rich-protein-genes-in-the-chinese-fire-bellied-newt-cynops-orientalis
#17
Yalong Feng, Juantao Feng, Hanxue Zheng, Wenjun Wang, Fulin Chen, Yuan Yu, Jihong Cui
The cysteine- and glycine-rich protein (CRP) family members, including the cysteine- and glycine-rich protein 1 (CSRP1), cysteine- and glycine-rich protein 2 (CSRP2), and the cysteine- and glycine-rich protein 3 (CSRP3), have exhibited various cellular functions during cell development and differentiation. However, the sequences of the three CSRP genes and their functions are still poorly understood in newts. In this study, we cloned the complete open reading frame (ORF) sequences of the three CSRP genes from the Chinese fire-bellied newt, Cynops orientalis (C...
January 6, 2018: Gene
https://www.readbyqxmd.com/read/29314423/kr%C3%A3-ppel-homologue-1-acts-as-a-repressor-and-an-activator-in-the-transcriptional-response-to-juvenile-hormone-in-adult-mosquitoes
#18
R Ojani, X Fu, T Ahmed, P Liu, J Zhu
Krüppel homologue 1 (Kr-h1) is a zinc finger transcription factor that is upregulated in insects by juvenile hormone (JH) in metamorphosis and adult reproduction. The molecular function of Kr-h1 in reproduction remains largely unknown. Here we report that AaKr-h1 functions as an important transcription regulator in adult female Aedes aegypti mosquitoes. The amount of AaKr-h1 protein increases with rising JH levels after adult emergence, reaches its peak at 48 h after eclosion, then decreases gradually and disappears after blood feeding...
January 4, 2018: Insect Molecular Biology
https://www.readbyqxmd.com/read/29313866/extensively-drug-resistant-arma-producing-acinetobacter-baumannii-in-an-italian-intensive-care-unit
#19
Carla Caio, Gaetano Maugeri, Tiziana Zingali, Floriana Gona, Stefania Stefani, Maria Lina Mezzatesta
We describe the spread of 12 carbapenem-resistant Acinetobacter baumannii isolates in hospitalized patients. All strains showed an extensively drug-resistant phenotype and high-level of aminoglycoside resistance, harboring the ArmA gene and blaoxa-23 downstream of ISAba1 (transposon Tn2008 arrangement) where both were located on the chromosome. These strains carry a class 1 integrone containing the gene cassette aacA4-catB8-aadA1. Molecular analysis revealed that all isolates belonged to the same sequence type (ST) 2 clone...
January 9, 2018: New Microbiologica
https://www.readbyqxmd.com/read/29312617/t-cell-responses-to-kshv-infection-a-systematic-approach
#20
Romin Roshan, Nazzarena Labo, Matthew Trivett, Wendell Miley, Vickie Marshall, Lori Coren, Elena M Cornejo Castro, Hannah Perez, Benjamin Holdridge, Eliza Davis, Rodrigo Matus-Nicodemos, Victor I Ayala, Raymond Sowder, Kathleen M Wyvill, Karen Aleman, Christine Fennessey, Jeffrey Lifson, Mark N Polizzotto, Daniel Douek, Brandon Keele, Thomas S Uldrick, Robert Yarchoan, Claes Ohlen, David Ott, Denise Whitby
Prior studies of T-cell responses to KSHV have included relatively few participants and focused on relatively few KSHV antigens. To provide a more comprehensive analysis, we investigated T-cell responses to the whole KSHV proteome using IFN-γ ELISpot. Using ∼7,500 overlapping 15mer peptides we generated one to three peptide pools for each of the 82 KSHV ORFs. IFN-γ ELISpot analysis of PBMCs from 19 patients with a history of KSHV-associated disease and 24 healthy donors (11 KSHV seropositive) detected widely varied responses...
December 12, 2017: Oncotarget
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