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Molecular Biotechnology

Liliane M Fernandes de Oliveira, Mirian G Morale, Agtha A M Chaves, Marilene Demasi, Paulo L Ho
Ubiquitin-proteasome system plays an essential role in the immune response due to its involvement in the antigen generation and presentation to CD8(+) T cells. Hereby, ubiquitin fused to antigens has been explored as an immunotherapeutic strategy that requires the activation of cytotoxic T lymphocytes. Here we propose to apply this ubiquitin fusion approach to a recombinant vaccine against human papillomavirus 16-infected cells. E6E7 multi-epitope antigen was fused genetically at its N- or C-terminal end to ubiquitin and expressed in Escherichia coli as inclusion bodies...
December 26, 2016: Molecular Biotechnology
Rahul Jain, Anita Pandey, Mukesh Pasupuleti, Veena Pande
Pseudomonas, being the common inhabitant of colder environments, are suitable for the production of cold-active enzymes. In the present study, a newly isolated strain of Pseudomonas from cold desert site in Indian Himalayan Region, was investigated for the production of cold-active lipase. The bacteria were identified as Pseudomonas proteolytica by 16S rDNA sequencing. Lipase production by bacteria was confirmed by qualitative assay using tributyrin and rhodamine-B agar plate method. The bacterium produced maximum lipase at 25 °C followed by production at 15 °C while utilizing olive, corn, as well as soybean oil as substrate in lipase production broth...
December 24, 2016: Molecular Biotechnology
Ronald E Hector, Jeffrey A Mertens
Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DNA-binding repressor (XylR) from Caulobacter crescentus was used to block transcription from synthetic promoters based on the constitutive Ashbya gossypii TEF promoter. The new hybrid promoters were repressed in the absence of xylose and showed up to a 25-fold increase in the presence of xylose...
December 23, 2016: Molecular Biotechnology
Evangelia Kontopoulou, Oumaima Stambouli, Sanjana Mathew
No abstract text is available yet for this article.
December 14, 2016: Molecular Biotechnology
Marc-André Robert, Viktoria Lytvyn, Francis Deforet, Rénald Gilbert, Bruno Gaillet
Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4)...
November 9, 2016: Molecular Biotechnology
Marcus Jenn Yang Chee, Grantley W Lycett, Teng-Jin Khoo, Chiew Foan Chin
Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively...
November 8, 2016: Molecular Biotechnology
Evangelia Kontopoulou, Oumaima Stambouli, Sanjana Mathew
No abstract text is available yet for this article.
December 2016: Molecular Biotechnology
Sandeep Kumar, Kavish Kr Jain, Shikha Rani, Kailash N Bhardwaj, Manisha Goel, Ramesh Chander Kuhad
Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C...
December 2016: Molecular Biotechnology
Rajib Deb, Gyanendra Singh Sengar, Umesh Singh, Sushil Kumar, R R Alyethodi, Rani Alex, T V Raja, A K Das, B Prakash
Loop-mediated isothermal amplification (LAMP) is a diagnostic method for amplification of DNA with rapid and minimal equipment requirement. In the present study, we applied the LAMP assay for rapid detection of cow components adulteration in buffalo milk/meat samples. The test can be completed within around 1 h 40 min starting from DNA extraction and can be performed in water bath without requirement of thermocycler. The cow DNA in buffalo samples were identified in the developed LAMP assay by either visualizing with SYBR Green I/HNB dyes or observing the typical ladder pattern on gel electrophoresis...
December 2016: Molecular Biotechnology
Aneela Yasmeen, Sarfraz Kiani, Afshan Butt, Abdul Qayyum Rao, Faheem Akram, Aftab Ahmad, Idrees Ahmad Nasir, Tayyab Husnain, Shahid Mansoor, Imran Amin, Shaheen Aftab, Muhammad Zubair, Muhammad Nouman Tahir, Sohail Akhtar, Jodi Scheffler, Brian Scheffler
The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively...
December 2016: Molecular Biotechnology
O Sakiragaoglu, A L Munn
To inhibit telomerase activity, a construct which contains artificial introns in the enhanced green fluorescent protein (EGFP) gene that encodes small hairpin RNA (shRNA) sequences that target human telomerase reverse transcriptase (hTERT) gene expression was designed and tested for its effect on lung cancer cell line. On intron splicing from the construct, intronic sequences were released and formed shRNA in the cells. After transfection of the construct, hTERT mRNA expression decreased by approximately 55 % in A549 cells...
December 2016: Molecular Biotechnology
Zifan Gong, Mackenzie T Walls, Alisha N Karley, Amy J Karlsson
Cell-penetrating peptides (CPPs) are a class of small peptides that are able to cross cell membranes via direct translocation or endocytosis. They have been widely used to deliver tethered bioactive molecules to cells, but recombinantly producing CPPs as fusions to protein cargo leads to low yields. We used Escherichia coli cells to recombinantly produce genetic fusions of NPFSD (derived from a yeast endocytosis signal) and pVEC (derived from a murine vascular endothelium cadherin) to the N-terminus of green fluorescent protein (GFP) with and without a flexible glycine-serine linker between the CPP and GFP...
December 2016: Molecular Biotechnology
Ana C Colabardini, Mari Valkonen, Anne Huuskonen, Matti Siika-Aho, Anu Koivula, Gustavo H Goldman, Markku Saloheimo
Two novel GH3 family thermostable β-glucosidases from the filamentous fungus Chaetomium atrobrunneum (CEL3a and CEL3b) were expressed in Trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. Both enzymes were active over a wide range of pH as compared to Neurospora crassa β-glucosidase GH3-3, which was also expressed in T. reesei and purified. The optimum temperature of both C. atrobrunneum enzymes was around 60 °C at pH 5, and both enzymes had better thermal and pH stability and higher resistance to metallic compounds and to glucose inhibition than GH3-3...
December 2016: Molecular Biotechnology
Sandun Fernando, Teshan Fernando, Michal Stefanik, Ludek Eyer, Daniel Ruzek
To find an effective drug for Zika virus, it is important to understand how numerous proteins which are critical for the virus' structure and function interact with their counterparts. One approach to inhibiting the flavivirus is to deter its ability to bind onto glycoproteins; however, the crystal structures of envelope proteins of the ever-evolving viral strains that decipher glycosidic or drug-molecular interactions are not always available. To fill this gap, we are reporting a holistic, simulation-based approach to predict compounds that will inhibit ligand binding onto a structurally unresolved protein, in this case the Zika virus envelope protein (ZVEP), by developing a three-dimensional general structure and analyzing sites at which ligands and small drug-like molecules interact...
December 2016: Molecular Biotechnology
Hevila Brognaro, Vitor Medeiros Almeida, Evandro Ares de Araujo, Vasily Piyadov, Maria Auxiliadora Morim Santos, Sandro Roberto Marana, Igor Polikarpov
The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C...
December 2016: Molecular Biotechnology
Somayeh Shatizadeh Malekshahi, Vahid Salimi, Ehsan Arefian, Ghazal Fatemi-Nasab, Sarvin Adjaminejad-Fard, Jila Yavarian, Talat Mokhtari-Azad
We attempted to generate siRNAs with two active strands, which can simultaneously knock down the expression of mRNA and viral genomic RNA. In this study, short hairpin RNAs (shRNAs) against N and F genes were used. Expression of F and N mRNA transcripts as well as genomic RNA was determined with relative real-time RT-PCR. The RSV load in infected cell culture supernatant was determined by absolute quantitative real-time PCR. We found that (i) in the presence of shRNA-N, a greater reduction in viral genomic RNA was found; (ii) the level of expression at MOI 0...
November 2016: Molecular Biotechnology
Huimin Zhang, Hongkui He, Xiujuan Yu, Zhaohui Xu, Zhizhou Zhang
It remains an unsolved problem to quantify a natural microbial community by rapidly and conveniently measuring multiple species with functional significance. Most widely used high throughput next-generation sequencing methods can only generate information mainly for genus-level taxonomic identification and quantification, and detection of multiple species in a complex microbial community is still heavily dependent on approaches based on near full-length ribosome RNA gene or genome sequence information. In this study, we used near full-length rRNA gene library sequencing plus Primer-Blast to design species-specific primers based on whole microbial genome sequences...
November 2016: Molecular Biotechnology
Gengtao Qiu, Ping Wang, Guangjun Li, Zhanjun Shi, Michael D Weir, Jinyu Sun, Yang Song, Jixing Wang, Huakun H Xu, Liang Zhao
Calcium phosphate cements (CPCs) are a new generation of bone repair materials with good biocompatibility for various stem cells. The minipig is a recommended large animal model for bone engineering research. This study aimed to evaluate the feasibility of utilizing CPC scaffolds for the adhesion, proliferation, and osteogenic differentiation of minipig's bone marrow mesenchymal stem cells (pBMSCs). Passage 3 pBMSCs were seeded on the CPC scaffold and cultured with osteogenic culture medium (osteogenic group) or normal medium (control group)...
November 2016: Molecular Biotechnology
F Mandelli, T A Gonçalves, C A Gandin, A C P Oliveira, M Oliveira Neto, F M Squina
Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80 °C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP)...
November 2016: Molecular Biotechnology
Roshank Ahani, Farzin Roohvand, Reza Ahangari Cohan, Mohammad Hossein Etemadzadeh, Nasir Mohajel, Mahdi Behdani, Zahra Shahosseini, Navid Madani, Kayhan Azadmanesh
Introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (LVs), is crucial in their systemic administration for cancer gene therapy. The pivotal role of tumor-associated endothelial cells (TAECs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (VEGFR2 or KDR) in TAECs makes them a potent target in cancer treatment. Herein, we report the development of VEGFR2-targeted LVs pseudotyped with chimeric sindbis virus E2 glycoprotein (cSVE2s)...
November 2016: Molecular Biotechnology
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