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https://www.readbyqxmd.com/read/28631643/multidrug-resistant-tuberculosis-detection-and-characterization-of-mutations-in-mycobacterium-tuberculosis-by-genotype-mtbdrplus
#1
Rajneesh Tripathi, Shampa Anupurba
Detection of drug resistance in Mycobacterium tuberculosis by conventional phenotypic drug susceptibility testing methods requires several weeks. Therefore, molecular diagnostic tests for rapid detection of multidrug resistance tuberculosis (MDR-TB) are urgently needed. Early diagnosis helps in initiating optimal treatment which would not only enable cure of an individual patient but also will curb the transmission of drug resistance in the community. Line probe assay (LPA) has shown great promises in the diagnosis of MDR-TB...
April 2017: Indian Journal of Pathology & Microbiology
https://www.readbyqxmd.com/read/28630449/effect-of-dna-sequence-of-fab-fragment-on-yield-characteristics-and-cell-growth-of-e-coli
#2
Antti Kulmala, Tuomas Huovinen, Urpo Lamminmäki
Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains...
June 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28630288/intact-pirna-pathway-prevents-l1-mobilization-in-male-meiosis
#3
Simon J Newkirk, Suman Lee, Fiorella C Grandi, Valeriya Gaysinskaya, James M Rosser, Nicole Vanden Berg, Cathryn A Hogarth, Maria C N Marchetto, Alysson R Muotri, Michael D Griswold, Ping Ye, Alex Bortvin, Fred H Gage, Jef D Boeke, Wenfeng An
The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter...
June 19, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28630112/rapid-evolution-of-ovarian-biased-genes-in-the-yellow-fever-mosquito-aedes-aegypti
#4
Carrie A Whittle, Cassandra G Extavour
Males and females exhibit highly dimorphic phenotypes, particularly in their gonads, which is believed to be driven largely by differential gene expression. Typically, the protein sequences of genes upregulated in males, or male-biased genes, evolve rapidly as compared to female-biased and unbiased genes. To date, the specific study of gonad-biased genes remains uncommon in metazoans. Here, we identified and studied a total of 2,927, 2,013 and 4,449 coding-sequences (CDS) with ovary-biased, testis-biased and unbiased expression respectively in the yellow-fever mosquito Aedes aegypti The results showed that ovary-biased and unbiased CDS had higher nonsynonymous to synonymous substitution rates (dN/dS) and lower optimal codon usage (those codons that promote efficient translation) than testis-biased genes...
June 19, 2017: Genetics
https://www.readbyqxmd.com/read/28629922/vaccination-with-a-codon-optimized-a27l-containing-plasmid-decreases-virus-replication-and-dissemination-after-vaccinia-virus-challenge
#5
Osmarie Martínez, Ariana Bravo Cruz, Saritza Santos, Maite Ramírez, Eric Miranda, Joanna Shisler, Miguel Otero
Smallpox is a disease caused by Variola virus (VARV). Although eradicated by WHO in 1980, the threat of using VARV on a bioterror attack has increased. The current smallpox vaccine ACAM2000, which consists of live vaccinia virus (VACV), causes complications in individuals with a compromised immune system or with previously reported skin diseases. Thus, a safer and efficacious vaccine needs to be developed. Previously, we reported that our virus-free DNA vaccine formulation, a pVAX1 plasmid encoding codon-optimized VACV A27L gene (pA27LOPT) with and without Imiquimod adjuvant, stimulates A27L-specific production of IFN-γ and increases humoral immunity 7days post-vaccination...
June 16, 2017: Vaccine
https://www.readbyqxmd.com/read/28627388/improved-soluble-bacterial-expression-and-properties-of-the-recombinant-flavonoid-glucosyltransferase-ugt73g1-from-allium-cepa
#6
Ruxin Cai, Caihong Chen, Yan Li, Kaiyan Sun, Fangfang Zhou, Kequan Chen, Honghua Jia
Glycosylation of quercetin using flavonol-specific glycosyltransferases offers an alternate method for isoquercitrin production. Obtaining sufficient quantities of bioactive enzymes is an important prerequisite for highly effective biocatalysis and biotransformation. In this study, a codon-optimized gene for the flavonoid glucosyltransferase UGT73G1 from Allium cepa was heterologously expressed in the preferred prokaryotic expression host Escherichia coli. By combining expression as a fusion protein with 6-histidine tags with coexpression with molecular chaperones, increased soluble expression of UGT73G1 was achieved in E...
June 13, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28625482/equalizing-the-information-amounts-of-protein-and-mrna-by-information-theory
#7
Y Adiguzel
Based on the Shannon's information communication theory, information amount of the entire length of a polymeric macromolecule can be calculated in bits through adding the entropies of each building block. Proteins, DNA and RNA are such macromolecules. When only the building blocks' variation is considered as the source of entropy, there is seemingly lower information in case of the protein if this approach is applied directly on a protein of specific size and the coding sequence size of the mRNA corresponding to the particular length of the protein...
June 15, 2017: Bio Systems
https://www.readbyqxmd.com/read/28624210/low-dose-gene-therapy-for-murine-pku-using-episomal-naked-dna-vectors-expressing-pah-from-its-endogenous-liver-promoter
#8
Hiu Man Grisch-Chan, Andrea Schlegel, Tanja Scherer, Gabriella Allegri, Raphael Heidelberger, Panagiota Tsikrika, Marco Schmeer, Martin Schleef, Cary O Harding, Johannes Häberle, Beat Thöny
Limited duration of transgene expression, insertional mutagenesis, and size limitations for transgene cassettes pose challenges and risk factors for many gene therapy vectors. Here, we report on physiological expression of liver phenylalanine hydroxylase (PAH) by delivery of naked DNA/minicircle (MC)-based vectors for correction of homozygous enu2 mice, a model of human phenylketonuria (PKU). Because MC vectors lack a defined size limit, we constructed a MC vector expressing a codon-optimized murine Pah cDNA that includes a truncated intron and is under the transcriptional control of a 3...
June 16, 2017: Molecular Therapy. Nucleic Acids
https://www.readbyqxmd.com/read/28620587/a-dual-luciferase-reporter-system-for-b-burgdorferi-measures-transcriptional-activity-during-tick-pathogen-interactions
#9
Philip P Adams, Carlos Flores Avile, Mollie W Jewett
Knowledge of the transcriptional responses of vector-borne pathogens at the vector-pathogen interface is critical for understanding disease transmission. Borrelia (Borreliella) burgdorferi, the causative agent of Lyme disease in the United States, is transmitted by the bite of infected Ixodes sp. ticks. It is known that B. burgdorferi has altered patterns of gene expression during tick acquisition, persistence and transmission. Recently, we and others have discovered in vitro expression of RNAs found internal, overlapping, and antisense to annotated open reading frames in the B...
2017: Frontiers in Cellular and Infection Microbiology
https://www.readbyqxmd.com/read/28607160/a-pria-mutant-expressed-in-two-pieces-has-almost-full-activity-in-e-coli-k-12
#10
Maxime Leroux, Niketa Jani, Steven J Sandler
The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC and dnaT genes encode the Replication Restart Proteins (RRPs) to accomplish this task. PriA plays a critical role in Replication Restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par(-)), UV sensitivity and recombination deficiency. PriA has 733 amino acids and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, re-model the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB and DnaT, and display ATPase, helicase and translocase activities...
June 12, 2017: Journal of Bacteriology
https://www.readbyqxmd.com/read/28605882/toward-a-cell-free-hydantoinase-process-screening-for-expression-optimization-and-one-step-purification-as-well-as-immobilization-of-hydantoinase-and-carbamoylase
#11
Christin Slomka, Georg Paris Späth, Phillip Lemke, Marc Skoupi, Christof M Niemeyer, Christoph Syldatk, Jens Rudat
The hydantoinase process is applied for the industrial synthesis of optically pure amino acids via whole cell biocatalysis, providing a simple and well-established method to obtain the catalyst. Nevertheless, whole cell approaches also bear disadvantages like intracellular degradation reactions, transport limitations as well as low substrate solubility. In this work the hydantoinase and carbamoylase from Arthrobacter crystallopoietes DSM 20117 were investigated with respect to their applicability in a cell-free hydantoinase process...
December 2017: AMB Express
https://www.readbyqxmd.com/read/28601219/the-importance-of-being-modified-the-role-of-rna-modifications-in-translational-fidelity
#12
Paul F Agris, Amithi Narendran, Kathryn Sarachan, Ville Y P Väre, Emily Eruysal
The posttranscriptional modifications of tRNA's anticodon stem and loop (ASL) domain represent a third level, a third code, to the accuracy and efficiency of translating mRNA codons into the correct amino acid sequence of proteins. Modifications of tRNA's ASL domain are enzymatically synthesized and site specifically located at the anticodon wobble position-34 and 3'-adjacent to the anticodon at position-37. Degeneracy of the 64 Universal Genetic Codes and the limitation in the number of tRNA species require some tRNAs to decode more than one codon...
2017: Enzymes
https://www.readbyqxmd.com/read/28596569/targeting-bacillus-anthracis-toxicity-with-a-genetically-selected-inhibitor-of-the-pa-cmg2-protein-protein-interaction
#13
Abigail L Male, Fedor Forafonov, Francesco Cuda, Gong Zhang, Siqi Zheng, Petra C F Oyston, Peng R Chen, E Diane Williamson, Ali Tavassoli
The protein-protein interaction between the human CMG2 receptor and the Bacillus anthracis protective antigen (PA) is essential for the transport of anthrax lethal and edema toxins into human cells. We used a genetically encoded high throughput screening platform to screen a SICLOPPS library of 3.2 million cyclic hexapeptides for inhibitors of this protein-protein interaction. Unusually, the top 3 hits all contained stop codons in the randomized region of the library, resulting in linear rather than cyclic peptides...
June 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28596531/rf1-attenuation-enables-efficient-non-natural-amino-acid-incorporation-for-production-of-homogeneous-antibody-drug-conjugates
#14
Gang Yin, Heather T Stephenson, Junhao Yang, Xiaofan Li, Stephanie M Armstrong, Tyler H Heibeck, Cuong Tran, Mary Rose Masikat, Sihong Zhou, Ryan L Stafford, Alice Y Yam, John Lee, Alexander R Steiner, Avinash Gill, Kalyani Penta, Sonia Pollitt, Ramesh Baliga, Christopher J Murray, Christopher D Thanos, Leslie M McEvoy, Aaron K Sato, Trevor J Hallam
Amber codon suppression for the insertion of non-natural amino acids (nnAAs) is limited by competition with release factor 1 (RF1). Here we describe the genome engineering of a RF1 mutant strain that enhances suppression efficiency during cell-free protein synthesis, without significantly impacting cell growth during biomass production. Specifically, an out membrane protease (OmpT) cleavage site was engineered into the switch loop of RF1, which enables its conditional inactivation during cell lysis. This facilitates extract production without additional processing steps, resulting in a scaleable extract production process...
June 8, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28591592/recombinant-thermo-alkali-stable-endoglucanase-of-myceliopthora-thermophila-bja-rmt-egl-biochemical-characteristics-and-applicability-in-enzymatic-saccharification-of-agro-residues
#15
Priya Phadtare, Swati Joshi, T Satyanarayana
Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould Myceliopthora thermophile BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (Mt-egl) has been constitutively expressed in Pichia pastoris under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t1/2 of 60 and 15min at 90 and 100°C, respectively...
June 4, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28590129/high-level-production-and-properties-of-the-cysteine-depleted-cytochrome-p450-3a4
#16
Irina F Sevrioukova
Human drug-metabolizing cytochrome P450 3A4 (CYP3A4) is a dynamic enzyme with a large and highly malleable active site that can fit structurally diverse compounds. Despite extensive investigations, structure-function relationships and conformational dynamics in CYP3A4 are not fully understood. This study was undertaken to engineer a well-expressed and functionally active cysteine-depleted CYP3A4 that can be used in biochemical and biophysical studies. cDNA codon optimization and screening mutagenesis were utilized to boost the level of bacterial expression of CYP3A4 and identify the least harmful substitutions for all six non-heme-ligating cysteines...
June 7, 2017: Biochemistry
https://www.readbyqxmd.com/read/28588310/the-development-of-fluorescent-protein-tracing-vectors-for-multicolor-imaging-of-clinically-isolated-staphylococcus-aureus
#17
Fuminori Kato, Motoki Nakamura, Motoyuki Sugai
Recent advances in fluorescent protein technology provide a wide variety of biological imaging applications; however current tools for bio-imaging in the Gram-positive bacterium Staphylococcus aureus has necessitated further developments for fluorescence intensity and for a multicolor palette of fluorescent proteins. To enhance the expression of multicolor fluorescent proteins in clinical S. aureus strains, we developed new fluorescent protein expression vectors, containing the blaZ/sodp promoter consisting of the β-lactamase gene (blaZ) promoter and the ribosome binding site (RBS) of superoxide dismutase gene (sod)...
June 6, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28582582/codon-usage-regulates-protein-structure-and-function-by-affecting-translation-elongation-speed-in-drosophila-cells
#18
Fangzhou Zhao, Chien-Hung Yu, Yi Liu
Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation...
June 5, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28579174/production-of-5-aminovaleric-acid-in-recombinant-corynebacterium-glutamicum-strains-from-a-miscanthus-hydrolysate-solution-prepared-by-a-newly-developed-miscanthus-hydrolysis-process
#19
Jeong Chan Joo, Young Hoon Oh, Ju Hyun Yu, Sung Min Hyun, Tae Uk Khang, Kyoung Hee Kang, Bong Keun Song, Kyungmoon Park, Min-Kyu Oh, Sang Yup Lee, Si Jae Park
This study examined nine expired industrial Corynebacterium glutamicum strains with high lysine producing capability for enhanced production of 5-AVA. C. glutamicum KCTC 1857 exhibiting the highest lysine production was transformed with either original Pseudomonas putida davBA genes, encoding the 5-AVA biosynthesis pathway, or C. glutamicum codon-optimized davBA genes. C. glutamicum KCTC 1857 expressing the original genes had superior cell viability and 5-AVA production capability compared to the other strain...
May 21, 2017: Bioresource Technology
https://www.readbyqxmd.com/read/28575317/ribosomal-selection-of-mrnas-with-degenerate-initiation-triplets
#20
He Chengguang, Paola Sabatini, Letizia Brandi, Anna M Giuliodori, Cynthia L Pon, Claudio O Gualerzi
To assess the influence of degenerate initiation triplets on mRNA recruitment by ribosomes, five mRNAs identical but for their start codon (AUG, GUG, UUG, AUU and AUA) were offered to a limiting amount of ribosomes, alone or in competition with an identical AUGmRNA bearing a mutation conferring different electrophoretic mobility to the product. Translational efficiency and competitiveness of test mRNAs toward this AUGmRNA were determined quantifying the relative amounts of the electrophoretically separated wt and mutated products synthesized in vitro and found to be influenced to different extents by the nature of their initiation triplet and by parameters such as temperature and nutrient availability in the medium...
May 27, 2017: Nucleic Acids Research
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