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Novel dna extraction

Riccardo Paolo Lia, Yasen Mutafchiev, Vincenzo Veneziano, Alessio Giannelli, Francesca Abramo, Mario Santoro, Maria Stefania Latrofa, Cinzia Cantacessi, Coralie Martin, Domenico Otranto, Andrea Bertuglia, Barbara Riccio
Equids can be infected by a range of skin-dwelling filarial nematodes, including four species of the genus Onchocerca. Current literature on equine onchocercosis is fragmentary and often limited to isolated case reports. The present study aimed to describe a clinical case of equine onchocercosis caused by Onchocerca boehmi (Supperer, 1953) (syn. Elaeophora boehmi) in an 8-year-old gelding Belgian show jumper from northern Italy. The horse was presented with a firm and painless mass on the proximal third of the right metacarpal region...
October 19, 2016: Parasitology Research
Victoria C Foletta, Michelle Palmieri, Joachim Kloehn, Shaun Mason, Stephen F Previs, Malcolm J McConville, Oliver M Sieber, Clinton R Bruce, Greg M Kowalski
Deuterated water (²H₂O), a stable isotopic tracer, provides a convenient and reliable way to label multiple cellular biomass components (macromolecules), thus permitting the calculation of their synthesis rates. Here, we have combined ²H₂O labelling, GC-MS analysis and a novel cell fractionation method to extract multiple biomass components (DNA, protein and lipids) from the one biological sample, thus permitting the simultaneous measurement of DNA (cell proliferation), protein and lipid synthesis rates...
October 13, 2016: Metabolites
Seong-Tshool Hong
The human intestine contains a massive and complex microbial community called gut microbiota. A typical human carries 100 trillion microbes in his/her body which is 10 times greater than the number of their host cells, i.e. whole number of human cells. A combined microbial genome constituting gut microbiota is well excess our own human genome. The microbial composition of gut microbiotata and its role on diseases became a booming area of research, presenting a new paradigm of opportunities for modern medicines...
September 2016: Journal of Hypertension
Yohei Kurosaki, Sayaka Okada, Sayuri Nakamae, Jiro Yasuda
Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction...
October 14, 2016: Journal of Virological Methods
Joshua J Meeks, Benedito A Carneiro, Sachin G Pai, Daniel T Oberlin, Alfred Rademaker, Kyle Fedorchak, Sohail Balasubramanian, Julia Elvin, Nike Beaubier, Francis J Giles
The genetic mechanisms associated with progression of high-risk non-muscle-invasive bladder cancer (HR-NMIBC) have not been described. We conducted selective next-generation sequencing (NGS) of HR-NMIBC and compared the genomic profiles of cancers that responded to intravesical therapy and those that progressed to muscle-invasive or advanced disease. DNA was extracted from paraffin-embedded sections from 25 HR-NMIBCs (22 with T1HG; 3 with TaHG with or without carcinoma in situ). Ten patients with HR-NMIBC developed progression (pT2+ or N+) ("progressors")...
October 14, 2016: Oncotarget
Jing Zhang, Chi Shu, Yanlin Song, Qingfang Li, Jingwen Huang, Xuelei Ma
BACKGROUND: The plasma Epstein-Barr virus (EBV) DNA level in patients with nasopharyngeal carcinoma (NPC) performs as an appealing prognostic factor, but conclusions of its prognostic values from previous studies are inconsistent. In this study, we performed a comprehensive meta-analysis to evaluate the prognostic value of EBV DNA level in patients with NPC. METHODS: Published studies were searched in PubMed. The baseline characteristics of patients, overall survival (OS), and other survival outcomes were extracted...
October 2016: Medicine (Baltimore)
Anders Breinbjerg, Charlotte Siggaard Rittig, Niels Gregersen, Søren Rittig, Jane Hvarregaard Christensen
AIM: Bartter syndrome is an autosomal recessive inherited disease in which patients present with hypokalaemia and metabolic alkalosis. We present two apparently non-related cases with antenatal Bartter syndrome type I, due to a novel variant in the SLC12A1 gene encoding the bumetanide-sensitive sodium-(potassium)-chloride cotransporter 2 in the thick ascending limb of the loop of Henle. METHODS: Blood samples were received from the two cases and 19 of their relatives and deoxyribonucleic acid was extracted...
October 17, 2016: Acta Paediatrica
Kim E M van Kessel, Willemien Beukers, Irene Lurkin, Angelique Ziel-van der Made, Kirstin A van der Keur, Joost L Boormans, Lars Dyrskjøt, Mirari Márquez, Torben F Ørntoft, Francisco X Real, Ulrika Segersten, Núria Malats, Per-Uno Malmström, Wim Van Criekinge, Ellen C Zwarthoff
PURPOSE: Only 3-28% of patients referred to the urology clinic for hematuria are diagnosed with bladder cancer (BC). Cystoscopies lead to high diagnostic costs and patient burden. Therefore, to improve selection of patients for cystoscopy, reduce costs and over-testing we aimed to validate a recently developed diagnostic urine assay MATERIALS AND METHODS: We included 200 patients from three European countries undergoing cystoscopy for hematuria (n=97 BC, n=103 non-malignant). Voided urine samples were collected prior to cystoscopy...
October 13, 2016: Journal of Urology
Yuko M Komesu, Holly E Richter, Darrell L Dinwiddie, Nazema Y Siddiqui, Vivian W Sung, Emily S Lukacz, Beri Ridgeway, Lily A Arya, Halina M Zyczynski, Rebecca G Rogers, Marie Gantz
INTRODUCTION AND HYPOTHESIS: We describe the rationale and methods of a study designed to compare vaginal and urinary microbiomes in women with mixed urinary incontinence (MUI) and similarly aged, asymptomatic controls. METHODS: This paper delineates the methodology of a supplementary microbiome study nested in an ongoing randomized controlled trial comparing a standardized perioperative behavioral/pelvic floor exercise intervention plus midurethral sling versus midurethral sling alone for MUI...
October 13, 2016: International Urogynecology Journal
Cerys J Evans, Alice E Davidson, Nicole Carnt, Karla E Rojas López, Neyme Veli, Caroline M Thaung, Stephen J Tuft, Alison J Hardcastle
Purpose: The majority of anterior corneal dystrophies are caused by dominant mutations in TGFBI (transforming growth factor β-induced) collectively known as the epithelial-stromal TGFBI dystrophies. Most cases of epithelial basement membrane dystrophy (EBMD) are thought to result from a degenerative (nongenetic) process; however, a minority of cases are associated with specific TGFBI mutations. We evaluated the spectrum of TGFBI mutations and associated phenotypes in a United Kingdom cohort with typical epithelial-stromal TGFBI dystrophies and an EBMD cohort...
October 1, 2016: Investigative Ophthalmology & Visual Science
Nan Chen, Jingying Sun, Yali Song, Xinjing Wei, Yan Shi, Li Zhang
BACKGROUND: Mutations of keratin 9 (KRT9) gene is a hot research area of epidermolytic palmoplantar keratoderma (EPPK). AIMS: To identify the genes caused the EPPK of a Chinese family. PATIENTS/METHODS: Three cases of lesions were collected for pathological examination. Genomic DNA was extracted from peripheral blood samples of six patients and five healthy individuals and 100 unrelated individuals. Polymerase chain reaction (PCR) was used to amplify exons 1 of KRT9 gene...
October 10, 2016: Journal of Cosmetic Dermatology
Hong Yan Liu, Jia Huang, Rui Li Wang, Yue Wang, Liang Jie Guo, Tao Li, Dong Wu, Hong Dan Wang, Qian Nan Guo, Dao Quan Dong
Familial exudative vitreoretinopathy (FEVR) is a hereditary ocular disorder characterized by a failure of peripheral retinal vascularization. In this report, we describe a novel missense mutation of the Norrie disease gene (NDP) in a Chinese family with X-linked FEVR. Ophthalmologic evaluation was performed on four male patients and seven unaffected individuals after informed consent was obtained. Venous blood was collected from the 11 members of this family, and genomic DNA was extracted using standard methods...
October 6, 2016: Journal of the Chinese Medical Association: JCMA
Ken Motohashi
Cell lysates from laboratory Escherichia coli strains endogenously exhibit homologous recombination activity, which can be utilized for seamless DNA cloning in vitro. This method, termed Seamless Ligation Cloning Extract (SLiCE) cloning, enables high cloning efficiency with simultaneous integration of two unpurified DNA fragments into a vector. In addition, the SLiCE method is highly cost-effective, as several laboratory E. coli strains may be utilized as sources of SLiCE. Previously, the SLiCE technique has been applied to site-directed mutagenesis to develop a novel technique termed SLiCE-mediated polymerase chain reaction (PCR)-based site-directed mutagenesis (SLiP site-directed mutagenesis)...
2017: Methods in Molecular Biology
Amar S Ahmad, Nataša Vasiljević, Paul Carter, Daniel M Berney, Henrik Møller, Christopher S Foster, Jack Cuzick, Attila T Lorincz
Clinically aggressive disease behavior is difficult to predict in men with low to intermediate clinical risk prostate cancer and methylation biomarkers may be a valuable adjunct for assessing the management of these patients. We set to evaluate the utility of DNA methylation to identify high risk disease in men currently considered as low or intermediate risk. DNA was extracted from formalin-fixed paraffin-embedded transurethral prostate resection tissues collected during the years 1990-96 in a watchful-waiting cohort of men in the UK...
September 30, 2016: Oncotarget
Samira Asgari, Paul J McLaren, Jane Peake, Melanie Wong, Richard Wong, Istvan Bartha, Joshua R Francis, Katia Abarca, Kyra A Gelderman, Philipp Agyeman, Christoph Aebi, Christoph Berger, Jacques Fellay, Luregn J Schlapbach
One out of three pediatric sepsis deaths in high income countries occur in previously healthy children. Primary immunodeficiencies (PIDs) have been postulated to underlie fulminant sepsis, but this concept remains to be confirmed in clinical practice. Pseudomonas aeruginosa (P. aeruginosa) is a common bacterium mostly associated with health care-related infections in immunocompromised individuals. However, in rare cases, it can cause sepsis in previously healthy children. We used exome sequencing and bioinformatic analysis to systematically search for genetic factors underpinning severe P...
2016: Frontiers in Immunology
Liang Zhang, Cuntao Yu, Qian Chang, Xinjin Luo, Juntao Qiu, Shen Liu
The aim of the present study was to compare the gene expression profiles in aortic dissection (AD) and healthy human aortic tissue samples by DNA microarray analysis in order to screen the differential genes. In total, five AD and four healthy aortic specimens were selected; the total RNA was extracted and reverse transcribed into cDNA and in vitro transcribed into aRNA, followed by microarray hybridization for analysis. Thereafter, the transcription levels of six differential genes, myosin light chain kinase (MYLK), polycystin 1, transient receptor potential channel interacting (PKD-1), myosin heavy chain 11 (MYH11), superoxide dismutase 3, extracellular (SOD3), filamin A (FLNA), and transgelin (TAGLN), screened from the expression profiles were quantitatively verified...
October 2016: Biomedical Reports
Amrita Panja, Prosanto Chowdhury, Anupam Basu
We describe a novel C > T substitution at codon 53 of the HBB gene (HBB: c.161C > T). The proband was a transfusion-dependent β-thalassemia major (β-TM) patient. DNA was extracted and subsequently, DNA sequencing was done to detect the mutations on the HBB gene. Capillary zone electrophoresis (CZE) revealed the presence of an unknown peak. She inherited this mutation from her grandmother through her mother. This mutation exists in cis with the common β(0) mutation IVS-I-5 (G > C) (HBB: c...
September 30, 2016: Hemoglobin
Juli-Anne Gardner, Jason D Peterson, Scott A Turner, Barbara L Soares, Courtney R Lancor, Luciana L Dos Santos, Prabhjot Kaur, Deborah L Ornstein, Gregory J Tsongalis, Francine B de Abreu
OBJECTIVES: To describe three methods used to screen for frameshift mutations in exon 9 of the CALR gene. METHODS: Genomic DNA from 47 patients was extracted from peripheral blood and bone marrow using the EZ1 DNA Blood Kit (Qiagen, Valencia, CA) and quantified by the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, San Diego, CA). After clinical history, cytogenetics, and molecular tests, patients were diagnosed with either clonal or nonclonal hematologic diseases...
October 2016: American Journal of Clinical Pathology
Anilkumar C Nirvanappa, Chakrabhavi Dhananjaya Mohan, Shobith Rangappa, Hanumappa Ananda, Alexey Yu Sukhorukov, Muthu K Shanmugam, Mahalingam S Sundaram, Siddaiah Chandra Nayaka, Kesturu S Girish, Arunachalam Chinnathambi, M E Zayed, Sulaiman Ali Alharbi, Gautam Sethi, Basappa, Kanchugarakoppal S Rangappa
Aberrant activation of nuclear factor kappa B (NF-κB) has been linked with the pathogenesis of several proinflammatory diseases including number of cancers and inflammatory bowel diseases. In the present work, we evaluated the anticancer activity of 1,2-oxazines derivatives against colorectal cancer cell lines and identified 2-((2-acetyl-6,6-dimethyl-4-phenyl-5,6-dihydro-2H-1,2-oxazin-3-yl)methyl)isoindoline-1,3-dione (API) as the lead anticancer agent among the tested compounds. The apoptosis inducing effect of API was demonstrated using flow cytometry analysis and measuring the caspase 3/7 activity in API treated cells...
2016: PloS One
Takeshi Shimoide, Naoyuki Kawao, Yukinori Tamura, Hironobu Morita, Hiroshi Kaji
Skeletal muscle hypertrophy and wasting are induced by hypergravity and microgravity, respectively. However, the mechanisms by which gravity change regulates muscle mass still remain unclear. We previously reported that hypergravity increases muscle mass via the vestibular system in mice. In this study, we performed comparative DNA microarray analysis of the soleus muscle from mice kept in 1 or 3 g environments with or without vestibular lesions. Mice were kept in 1 g or 3 g environment for 4 weeks by using a centrifuge 14 days after surgical bilateral vestibular lesions...
October 21, 2016: Biochemical and Biophysical Research Communications
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