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Molecular Biotechnology

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https://www.readbyqxmd.com/read/28526937/molecular-confirmation-of-intraspecific-tomato-solanum-lycopersicum-hybrids-and-their-evaluation-against-late-blight-and-cucumber-mosaic-virus
#1
Amjad Hameed, Muhammad Yussouf Saleem, Khalid Pervaiz Akhtar, Muhammad Shoaib, Qumer Iqbal, Muhammad Asghar
Tomato is one of the most consumed vegetables in the world. Diseases are the number one concern in the development of high-yield and disease-resistant tomato hybrids which is the foremost priority of breeders. Present study was conducted (1) to develop DNA-based markers for genetic confirmation of tomato F1 hybrids, (2) to utilize sequenced characterized amplified region (SCAR) marker linked to the Ph-3 gene for Phytophthora infestans resistance in tomato and (3) to evaluate male and female parental genotypes and their F1 hybrids against late blight (LB) and cucumber mosaic virus (CMV)...
May 19, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28509990/transient-activation-of-reprogramming-transcription-factors-using-protein-transduction-facilitates-conversion-of-human-fibroblasts-toward-cardiomyocyte-like-cells
#2
Zaniar Ghazizadeh, Hassan Rassouli, Hananeh Fonoudi, Mehdi Alikhani, Mahmood Talkhabi, Amir Darbandi-Azar, Shuibing Chen, Hossein Baharvand, Nasser Aghdami, Ghasem Hosseini Salekdeh
Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine)...
May 16, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28500482/improved-yield-of-high-molecular-weight-hyaluronic-acid-production-in-a-stable-strain-of-streptococcus-zooepidemicus-via-the-elimination-of-the-hyaluronidase-encoding-gene
#3
Navid Pourzardosht, Mohammad Javad Rasaee
Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana(r)-R as a recombinant suicide plasmid...
May 12, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28484957/characterization-of-recombinant-thermococcus-kodakaraensis-kod-dna-polymerases-produced-using-silkworm-baculovirus-expression-vector-system
#4
Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee
The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively...
May 8, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28447263/design-of-a-lentiviral-vector-for-the-inducible-expression-of-myc-a-new-strategy-for-construction-approach
#5
Onur Tokgun, Francesco Paolo Fiorentino, Pervin Elvan Tokgun, Jun Yokota, Hakan Akca
Lentiviral vectors are powerful tools for gene expression studies. Here we report the construction of pTIJ, a vector for inducible gene expression. pTIJ was generated from pTRIPZ backbone, which is designed for the inducible expression of shRNA sequences, by the introducing of a multiple cloning site upstream of the Tet promoter and the removal of miR30 flanking sequences. To evaluate pTIJ as a tool for the inducible expression of genes of interest, we introduced MYC cDNA into pTIJ and infected two small cell lung cancer cell lines, H209 and H345...
April 26, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28421327/metal-dna-interactions-improve-signal-in-high-resolution-melting-of-dna-for-species-differentiation-of-plasmodium-parasite
#6
Priyamvada Jain, Babina Chakma, Naveen Singh, Sanjukta Patra, Pranab Goswami
The success of high-resolution melting (HRM) analysis for distinguishing similar DNAs with minor base mismatch differences is limited. Here, metal-mediated structural change in DNA has been exploited to amplify HRM signals leading to differentiation of target DNAs in an orthologous gene corresponding to four Plasmodium species. Conserved 26-mer ssDNAs from ldh gene of the four Plasmodium species were employed as targets. A capture probe (CP) that is fully complementary to the Plasmodium falciparum target (FT) and has two base mismatches each, with the targets of Plasmodium vivax (VT), Plasmodium malariae, (MT), and Plasmodium ovale (OT), was considered...
April 18, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28374116/designing-an-escherichia-coli-strain-for-phenylalanine-overproduction-by-metabolic-engineering
#7
Neetu Tyagi, Deepti Saini, Richa Guleria, Krishna Jyoti Mukherjee
The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG (fbr) performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications...
April 3, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28349302/a-mutant-sumo-facilitates-quick-plasmid-construction-for-expressing-proteins-with-native-n-termini-after-tag-removal
#8
Yuzhu Zhang, Yuting Fan
Sumo is one of the fusion tags commonly used to enhance the expression and the solubility of recombinant proteins. One advantage of using sumo is that the removal of the sumo tag is highly specific because its recognition by a sumo protease is determined by its structural characteristics, instead of the sequence of a short peptide. Recently, it was reported that sumo could also be used as a protease recognition site to facilitate the removal of other fusion tags, such as MBP, when sumo itself is not suitable to enhance the solubility of a particular target protein...
March 27, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28342150/expression-and-characterization-of-human-%C3%AE-1-4-galactosyltransferase-1-%C3%AE-4galt1-using-silkworm-baculovirus-expression-system
#9
Daisuke Morokuma, Jian Xu, Masato Hino, Hiroaki Mon, Jasmeen S Merzaban, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee
Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm-BEVS...
March 24, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28342149/identification-and-preparation-of-a-novel-chemokine-receptor-binding-domain-in-the-cytoplasmic-regulator-frount
#10
Akihiro Sonoda, Sosuke Yoshinaga, Kaori Yunoki, Soichiro Ezaki, Kotaro Yano, Mitsuhiro Takeda, Etsuko Toda, Yuya Terashima, Kouji Matsushima, Hiroaki Terasawa
FROUNT is a cytoplasmic protein that binds to the membrane-proximal C-terminal regions (Pro-Cs) of chemokine receptors, CCR2 and CCR5. The FROUNT-chemokine receptor interactions play a pivotal role in the migration of inflammatory immune cells, indicating the potential of FROUNT as a drug target for inflammatory diseases. To provide the foundation for drug development, structural information of the Pro-C binding region of FROUNT is desired. Here, we defined the novel structural domain (FNT-CB), which mediates the interaction with the chemokine receptors...
March 24, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28332030/molecular-cloning-expression-and-characterization-of-pectin-methylesterase-ctpme-from-clostridium-thermocellum
#11
Vikky Rajulapati, Arun Goyal
Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells...
March 22, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28324209/molecular-cloning-structural-modeling-and-the-production-of-soluble-triple-mutated-diphtheria-toxoid-k51e-g52e-e148k-co-expressed-with-molecular-chaperones-in-recombinant-escherichia-coli
#12
Naphatsamon Uthailak, Pornpimol Mahamad, Pamorn Chittavanich, Somchai Yanarojana, Wassana Wijagkanalan, Jean Petre, Watanalai Panbangred
CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97...
March 21, 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28271340/an-overview-on-the-enhancement-of-enantioselectivity-and-stability-of-microbial-epoxide-hydrolases
#13
REVIEW
Priya Saini, Dipti Sareen
Epoxide hydrolases (EHs; 3.3.2.x) catalyze the enantioselective ring opening of racemic epoxides to the corresponding enantiopure vicinal diols and remaining equivalent unreacted epoxides. These epoxides and diols are used for the synthesis of chiral drug intermediates. With an upsurge in the methods for identification of novel microbial EHs, a lot of EHs have been discovered and utilized for kinetic resolution of racemic epoxides. However, there is still a constraint on the account of limited EHs being successfully applied on the preparative scale for industrial biotransformations...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28197768/expression-of-an-acid-urease-with-urethanase-activity-in-e-coli-and-analysis-of-urease-gene
#14
REVIEW
Xiaofeng Liu, Qian Zhang, Nandi Zhou, Yaping Tian
Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28194691/rnai-mediated-simultaneous-resistance-against-three-rna-viruses-in-potato
#15
Amir Hameed, Muhammad Nouman Tahir, Shaheen Asad, Rakhshanda Bilal, Joyce Van Eck, Georg Jander, Shahid Mansoor
RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28138902/ectopic-expression-of-plant-rna-chaperone-offering-multiple-stress-tolerance-in-e-coli
#16
Bushra Jabeen, S M Saqlan Naqvi, Tariq Mahmood, Tasawar Sultana, Madiha Arif, Fariha Khan
Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28132389/an-improved-method-for-identifying-specific-dna-protein-binding-sites-in-vitro
#17
Liangyan Wang, Huizhi Lu, Yunguang Wang, Su Yang, Hong Xu, Kaiying Cheng, Ye Zhao, Bing Tian, Yuejin Hua
Binding of proteins to specific DNA sequences is essential for a variety of cellular processes such as DNA replication, transcription and responses to external stimuli. Chromatin immunoprecipitation is widely used for determining intracellular DNA fragments bound by a specific protein. However, the subsequent specific or accurate DNA-protein-binding sequence is usually determined by DNA footprinting. Here, we report an alternative method for identifying specific sites of DNA-protein-binding (designated SSDP) in vitro...
March 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28025776/expression-polyubiquitination-and-therapeutic-potential-of-recombinant-e6e7-from-hpv16-antigens-fused-to-ubiquitin
#18
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...
January 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28013401/prolonged-production-and-aggregation-complexity-of-cold-active-lipase-from-pseudomonas-proteolytica-gbpi_hb61-isolated-from-cold-desert-himalaya
#19
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...
January 2017: Molecular Biotechnology
https://www.readbyqxmd.com/read/28012062/a-synthetic-hybrid-promoter-for-xylose-regulated-control-of-gene-expression-in-saccharomyces-yeasts
#20
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...
January 2017: Molecular Biotechnology
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