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BMC Biotechnology

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https://www.readbyqxmd.com/read/29764417/upf1-silenced-cellular-model-systems-for-screening-of-read-through-agents-active-on-%C3%AE-0-39-thalassemia-point-mutation
#1
Francesca Salvatori, Mariangela Pappadà, Giulia Breveglieri, Elisabetta D'Aversa, Alessia Finotti, Ilaria Lampronti, Roberto Gambari, Monica Borgatti
BACKGROUND: Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons...
May 15, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29751749/engineering-a-vitamin-b-12-high-throughput-screening-system-by-riboswitch-sensor-in-sinorhizobium-meliloti
#2
Yingying Cai, Miaomiao Xia, Huina Dong, Yuan Qian, Tongcun Zhang, Beiwei Zhu, Jinchuan Wu, Dawei Zhang
BACKGROUND: As a very important coenzyme in the cell metabolism, Vitamin B12 (cobalamin, VB12 ) has been widely used in food and medicine fields. The complete biosynthesis of VB12 requires approximately 30 genes, but overexpression of these genes did not result in expected increase of VB12 production. High-yield VB12 -producing strains are usually obtained by mutagenesis treatments, thus developing an efficient screening approach is urgently needed. RESULT: By the help of engineered strains with varied capacities of VB12 production, a riboswitch library was constructed and screened, and the btuB element from Salmonella typhimurium was identified as the best regulatory device...
May 11, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29743047/cloning-and-characterization-of-enoate-reductase-with-high-%C3%AE-ionone-to-dihydro-%C3%AE-ionone-bioconversion-productivity
#3
Xuesong Zhang, Shiyong Liao, Fuliang Cao, Linguo Zhao, Jianjun Pei, Feng Tang
BACKGROUND: Dihydro-β-ionone is a principal aroma compound and has received considerable attention by flavor and fragrance industry. The traditional method of preparing dihydro-β-ionone has many drawbacks, which has restricted its industrial application. Therefore, it is necessary to find a biotechnological method to produce dihydro-β-ionone. RESULTS: In this study, the enoate reductase with high conversion efficiency of β-ionone to dihydro-β-ionone, DBR1, was obtained by screening four genetically engineered bacteria...
May 9, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29728076/effective-preparation-of-a-monoclonal-antibody-against-human-chromogranin-a-for-immunohistochemical-diagnosis
#4
Danping Zhang, Chengjie Xie, Rongzhi Wang, Qinghai Yang, Huiling Chen, Sumei Ling, Shihua Wang, Kunzhi Jia
BACKGROUND: Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. RESULTS: The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice...
May 4, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29720159/development-of-a-baculovirus-vector-carrying-a-small-hairpin-rna-for-suppression-of-sf-caspase-1-expression-and-improvement-of-recombinant-protein-production
#5
Xiaoyue Zhang, Keyan Xu, Yanmei Ou, Xiaodong Xu, Hongying Chen
BACKGROUND: The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. RESULTS: In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector...
May 2, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29716562/heavy-ion-mutagenesis-combined-with-triclosan-screening-provides-a-new-strategy-for-improving-the-arachidonic-acid-yield-in-mortierella-alpina
#6
Huidan Zhang, Dong Lu, Xin Li, Yingang Feng, Qiu Cui, Xiaojin Song
BACKGROUND: Arachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment. RESULTS: The obtained mutant strain F-23 ultimately achieved an ARA yield of 5...
May 2, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29642881/antiplasmodial-and-trypanocidal-activity-of-violacein-and-deoxyviolacein-produced-from-synthetic-operons
#7
Elizabeth Bilsland, Tatyana A Tavella, Renata Krogh, Jamie E Stokes, Annabelle Roberts, James Ajioka, David R Spring, Adriano D Andricopulo, Fabio T M Costa, Stephen G Oliver
BACKGROUND: Violacein is a deep violet compound that is produced by a number of bacterial species. It is synthesized from tryptophan by a pathway that involves the sequential action of 5 different enzymes (encoded by genes vioA to vioE). Violacein has antibacterial, antiparasitic, and antiviral activities, and also has the potential of inducing apoptosis in certain cancer cells. RESULTS: Here, we describe the construction of a series of plasmids harboring the complete or partial violacein biosynthesis operon and their use to enable production of violacein and deoxyviolacein in E...
April 11, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29621998/a-factor-xia-activatable-hirudin-albumin-fusion-protein-reduces-thrombosis-in-mice-without-promoting-blood-loss
#8
William P Sheffield, Louise J Eltringham-Smith, Varsha Bhakta
BACKGROUND: Hirudin is a potent thrombin inhibitor but its antithrombotic properties are offset by bleeding side-effects. Because hirudin's N-terminus must engage thrombin's active site for effective inhibition, fusing a cleavable peptide at this site may improve hirudin's risk/benefit ratio as a therapeutic agent. Previously we engineered a plasmin cleavage site (C) between human serum albumin (HSA) and hirudin variant 3 (HV3) in fusion protein HSACHV3. Because coagulation factor XI (FXI) is more involved in thrombosis than hemostasis, we hypothesized that making HV3 activity FXIa-dependent would also improve HV3's potential therapeutic profile...
April 5, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29615011/soluble-expression-of-an-amebic-cysteine-protease-in-the-cytoplasm-of-escherichia-coli-shuffle-express-cells-and-purification-of-active-enzyme
#9
Ekaterina Jalomo-Khayrova, Rosa E Mares, Patricia L A Muñoz, Samuel G Meléndez-López, Ignacio A Rivero, Marco A Ramos
BACKGROUND: Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds...
April 3, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29606116/successful-production-of-genome-edited-rats-by-the-rgonad-method
#10
Tomoe Kobayashi, Masumi Namba, Takayuki Koyano, Masaki Fukushima, Masahiro Sato, Masato Ohtsuka, Makoto Matsuyama
BACKGROUND: Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as "improved GONAD" (i-GONAD). However, this technology has been limited only to mice...
April 2, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29558934/high-yield-production-and-purification-of-two-recombinant-thermostable-phosphotriesterase-like-lactonases-from-sulfolobus-acidocaldarius-and-sulfolobus-solfataricus-useful-as-bioremediation-tools-and-bioscavengers
#11
Odile Francesca Restaino, Maria Giovanna Borzacchiello, Ilaria Scognamiglio, Luigi Fedele, Alberto Alfano, Elena Porzio, Giuseppe Manco, Mario De Rosa, Chiara Schiraldi
BACKGROUND: Thermostable phosphotriesterase-like lactonases (PLLs) are able to degrade organophosphates and could be potentially employed as bioremediation tools and bioscavengers. But nowadays their manufacturing in high yields is still an issue that limits their industrial applications. In this work we aimed to set up a high yield production and purification biotechnological process of two recombinant PLLs expressed in E. coli, the wild type SacPox from Sulfolobus acidocaldarius and a triple mutated SsoPox C258L/I261F/W263A, originally from Sulfolobus solfataricus...
March 20, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29554888/cell-based-reference-samples-designed-with-specific-differences-in-microrna-biomarkers
#12
P Scott Pine, Steven P Lund, Sanford A Stass, Debra Kukuruga, Feng Jiang, Lynn Sorbara, Sudhir Srivastava, Marc Salit
BACKGROUND: We demonstrate the feasibility of creating a pair of reference samples to be used as surrogates for clinical samples measured in either a research or clinical laboratory setting. The reference sample paradigm presented and evaluated here is designed to assess the capability of a measurement process to detect true differences between two biological samples. Cell-based reference samples can be created with a biomarker signature pattern designed in silico. Clinical laboratories working in regulated applications are required to participate in proficiency testing programs; research laboratories doing discovery typically do not...
March 20, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29548320/total-rna-extraction-from-tissues-for-microrna-and-target-gene-expression-analysis-not-all-kits-are-created-equal
#13
Rikki A M Brown, Michael R Epis, Jessica L Horsham, Tasnuva D Kabir, Kirsty L Richardson, Peter J Leedman
BACKGROUND: microRNAs (miRNAs) are short non-coding RNAs that fine-tune gene expression. The aberrant expression of miRNAs is associated with many diseases and they have both therapeutic and biomarker potential. However, our understanding of their usefulness is dependent on the tools we have to study them. Previous studies have identified the need to optimise and standardise RNA extraction methods in order to avoid biased results. Herein, we extracted RNA from murine lung, liver and brain tissues using five commercially available total RNA extraction methods...
March 16, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29544494/phenotype-specific-recombinant-haptoglobin-polymers-co-expressed-with-c1r-like-protein-as-optimized-hemoglobin-binding-therapeutics
#14
Christian A Schaer, Catherine Owczarek, Jeremy W Deuel, Stefan Schauer, Jin Hyen Baek, Ayla Yalamanoglu, Matthew P Hardy, Pierre D Scotney, Peter M Schmidt, Matthias Pelzing, Peter Soupourmas, Paul W Buehler, Dominik J Schaer
BACKGROUND: Preclinical studies have evaluated haptoglobin (Hp) polymers from pooled human plasma as a therapeutic protein to attenuate toxic effects of cell-free hemoglobin (Hb). Proof of concept studies have demonstrated efficacy of Hp in hemolysis associated with transfusion and sickle cell anemia. However, phenotype-specific Hp products might be desirable to exploit phenotype specific activities of Hp 1-1 versus Hp 2-2, offering opportunities for recombinant therapeutics. Prohaptoglobin (proHp) is the primary translation product of the Hp mRNA...
March 15, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29540167/maintenance-of-human-chondrogenic-phenotype-on-a-dendrimer-immobilized-surface-for-an-application-of-cell-sheet-engineering
#15
Sopita Wongin, Saranatra Waikakul, Pojchong Chotiyarnwong, Wanwipa Siriwatwechakul, Masahiro Kino-Oka, Mee-Hae Kim, Kwanchanok Viravaidya-Pasuwat
BACKGROUND: Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets...
March 14, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29530017/microencapsulation-extends-mycelial-viability-of-streptomyces-lividans-66-and-increases-enzyme-production
#16
Boris Zacchetti, Agathoklis Andrianos, Dino van Dissel, Evelien de Ruiter, Gilles P van Wezel, Dennis Claessen
BACKGROUND: Filamentous bacteria of the genus Streptomyces produce a large arsenal of industrially relevant antibiotics and enzymes. The industrial production of these molecules occurs in large fermenters, where many streptomycetes form dense mycelial networks called pellets. Pellets are characterized by slow growth and inefficient nutrient transfer and therefore regarded as undesirable from the perspective of productivity. Although non-pelleting strains have increased growth rates, their morphology also leads to a dramatic increase in the viscosity of the culture broth, which negatively impacts the process dynamics...
March 12, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29514614/novel-hemostatic-biomolecules-based-on-elastin-like-polypeptides-and-the-self-assembling-peptide-rada-16
#17
Shasha Yang, Sili Wei, Yun Mao, Hanxue Zheng, Juantao Feng, Jihong Cui, Xin Xie, Fulin Chen, Honmgmin Li
BACKGROUND: Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination. RESULTS: The fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation...
March 7, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29454346/agrobacterium-mediated-and-electroporation-mediated-transformation-of-chlamydomonas-reinhardtii-a-comparative-study
#18
Paola Mini, Olivia Costantina Demurtas, Silvia Valentini, Patrizia Pallara, Giuseppe Aprea, Paola Ferrante, Giovanni Giuliano
BACKGROUND: Chlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time. RESULTS: We constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene...
February 17, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29439688/effects-of-substrate-binding-site-residue-substitutions-of-xyna-from-bacillus-amyloliquefaciens-on-substrate-specificity
#19
Anil S Prajapati, Vishakha A Pawar, Ketankumar J Panchal, Ankit P Sudhir, Bhaumik R Dave, Darshan H Patel, R B Subramanian
BACKGROUND: The aromatic residues of xylanase enzyme, W187, Y124, W144, Y128 and W63 of substrate binding pocket from Bacillus amyloliquefaciens were investigated for their role in substrate binding by homology modelling and sequence analysis. These residues are highly conserved and play an important role in substrate binding through steric hindrance. The substitution of these residues with alanine allows the enzyme to accommodate nonspecific substrates. RESULTS: Wild type and mutated genes were cloned and overexpressed in BL21...
February 13, 2018: BMC Biotechnology
https://www.readbyqxmd.com/read/29439686/genetically-encoded-calcium-indicator-with-ntnc-like-design-and-enhanced-fluorescence-contrast-and-kinetics
#20
D A Doronin, N V Barykina, O M Subach, V P Sotskov, V V Plusnin, O A Ivleva, E A Isaakova, A M Varizhuk, G E Pozmogova, A Y Malyshev, I V Smirnov, K D Piatkevich, K V Anokhin, G N Enikolopov, F V Subach
BACKGROUND: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2+ -binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-Ca2+ /F+Ca2+ ) and slow Ca2+ dissociation kinetics...
February 13, 2018: BMC Biotechnology
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