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Methods of Evaluation in Orthopaedic Animal Research
Published in Yuehuei H. An, Richard J. Friedman, Animal Models in Orthopaedic Research, 2020
Several methods are available for localization of specific genes, translocated chromosomes, or chromosomal breakpoints, for example: fluorescent in situ hybridization (FISH), in which a fluo-rescently labeled probe is allowed to hybridize to specific chromosomes and thus determines their gene location microscopically.252 Also, in situ hybridization (ISH) is capable of detecting mRNA for a specific gene, using nucleic acid probes which are complementary to and hybridize with the mRNA. Because it is performed on tissue sections, ISH localizes the mRNA to individual cells within the tissue and thereby allows investigation of the spatial distribution and heterogeneity of expression of particular genes within a population of cells. The method described by Hicks et al.253 is a typical example of ISH. It describes, in detail, a method for performing ISH on skeletal tissue cells (growth plate cells) using synthetic oligonucleotide (a short sequence of nucleotides) probes. This technique is currently being translated into diagnostic practice.254
Human Erythroenzymopathies Of The Anaerobic Embden-Meyerhof Glycolytic And Associated Pathways
Published in Ronald L. Nagel, Genetically Abnormal Red Cells, 2019
Ernst R. Jaffé, William N. Valentine
Information derived from careful biochemical examinations of the erythrocytes of patients with hereditary enzymopathies provides important and interesting insights into the pathophysiology of human disease. Not only have these studies led to the satisfaction of making accurate diagnoses in cases of unexplained hemolytic disorders, but they have identified the chromosome and even the gene location of the altered molecular code for the aberrant enzyme in many instances. These investigations are facilitated by the ease of obtaining the appropriate materials by repeated biopsies, i.e., venipuncture, and the ability to isolate pure preparations of erythrocytes. Increasingly, diagnostic clinical laboratories, aided by specialized research facilities, are able to report the data necessary to establish the diagnosis of well-recognized enzymopathies. The association of hemolysis with multisystem disease has also pointed the way to the diagnosis of obscure disorders whose clinical features result from a single molecular lesion. Thus, the findings highlight differences in the expression of abnormalities in different tissues and are most helpful in enhancing understanding of normal metabolic functions and regulations.
Overview of the Pharmacokinetics of Antiepileptic Drugs
Published in Carl L. Faingold, Gerhard H. Fromm, Drugs for Control of Epilepsy:, 2019
Genetics plays a role in the metabolism of phenytoin and mephenytoin.6,12,13 Genetically slow metabolizers of these drugs become overdosed with usual doses and need considerably smaller doses than average or fast metabolizers. The measurable parameters show low Vmax while Km is not changed considerably. The defects are autosomal recessive mediated by two alleles on the same gene location. These defects are analogous to but not identical with slow metabolism of debrisoquin and spartane.14
Stargardt disease and progress in therapeutic strategies
Published in Ophthalmic Genetics, 2022
Di Huang, Rachael C. Heath Jeffery, May Thandar Aung-Htut, Samuel McLenachan, Sue Fletcher, Steve D. Wilton, Fred K. Chen
Several groups refined the ABCA4 gene location to a 2–3 cM interval (36,37). Allikmets et al. localized two markers that flank the ABCA4 gene, D1S3361 and D1S236, to 1p22.3–1p22.2, therefore repositioning the gene to 1p22 (82). By fluorescence in situ hybridization, the chromosomal position of the human ABCA4 gene was mapped to 1p21-p22.1 (83), the current location published on the National Centre for Biotechnology Information website (NCBI, https://www.ncbi.nlm.nih.gov/nuccore/NM_000350.3). The coding region of the ABCA4 gene consists of 50 exons, spanning over 100 kb (39,82). According to Ensembl (http://www.ensembl.org), the ABCA4 exons range in size from 33 bp to 266 bp (Figure 4). There are 8 splice variants for ABCA4, three of which are protein-coding transcripts whilst the remaining are non-protein-coding. The full length, annotated ABCA4 transcript of 7328 bp, consisting of an open reading frame of 6822 bp encoding a 2273-amino-acid protein is published by NCBI (NP_000341.2).
Characterizing the gut microbiota in patients with chronic kidney disease
Published in Postgraduate Medicine, 2020
Xiaofang Hu, Shaxi Ouyang, Yuhong Xie, Zhicheng Gong, Jie Du
A region approximately 460 bp from the 16 S rRNA gene, designated as the V3-V4 region, was selected as a target gene location to construct the sequencing library. The common conserved primers, 341 F (5′-CCTACGGGNGGCWGCAG-3′) and 806 R (5′-GGACTACHVGGGTATCTAAT −3′), were used to amplify the target region. In addition, these primers contained a barcode sequence that allowed the analysis of multiple samples in a single sequencing run. PCR amplification was carried out in a 50 μL reaction system containing 5 μL of 10× KOD buffer, 5 μL of 2.5 mM dNTPs, 1.5 μL of 5 μM primers, 1 μL of KOD polymerase, and 100 ng of template DNA. PCRs were performed using the following cycle conditions: predenaturation at 95°C for 2 min, followed by 27 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 30 s, extension at 68°C for 30 s, and a final extension at 68°C for 10 min. Prior to sequencing, the DNA concentration of each PCR product was determined using a Quant-iTPico green double-stranded DNA assay (Invitrogen, California, USA) and its quality was assessed on an Agilent 2100 bioanalyzer (Agilent, USA). After several steps of purification, adaptor addition, and cDNA length selection, the libraries were used for sequencing on an Illumina HiSeq 2500 platform by Gene Denovo Biotechnology. (Guangzhou, China).
Down-expression of P2RX2, KCNQ5, ERBB3 and SOCS3 through DNA hypermethylation in elderly women with presbycusis
Published in Biomarkers, 2018
Amal Bouzid, Ibtihel Smeti, Leila Dhouib, Magali Roche, Imen Achour, Aida Khalfallah, Abdullah Ahmed Gibriel, Ilhem Charfeddine, Hammadi Ayadi, Joel Lachuer, Abdelmonem Ghorbel, Christine Petit, Saber Masmoudi
The raw sequencing reads were combined and analyzed using Methyl-seq analysis with Avadis NGS software v2.1 (Strand Life Sciences, Bangalore, India). DNA methylation analysis was performed at different levels as follows: (i) methylation detection in the two strand reads where both ends mapped uniquely in the correct orientation were combined for methylation level calculations. Bisulfite conversion error rate was estimated to be 0.1% for all samples and was computed by the External Standard method (Lister et al. 2009). The threshold parameters for detecting methylation sites were determined by: minimum read coverage: 10 and minimum base quality: 30. (ii) Differentially methylated cytosines (DMCs) detection was performed by comparing affected and control samples. (iii) Analysis of differentially methylated regions (DMRs) was conducted using sliding windows approach. For the successful identification of DMRs, we set up the following parameters: a sliding window of 500 bp minimum length that contains at least 10 DMCs (Fang et al. 2012) with sufficient coverage for calculation of DMC density (the number of DMCs divided by the number of methylated Cs) and to have statistical significance according to Fisher’s combined probability test with p-value cut off = 0.01. (iv) Annotation of the resulted DMCs was performed to identify DMR-related genes based on gene location information from UCSC gene annotations (hg19). The genes with at least one element (including promoter, 5′-UTR, exon, intron and 3′-UTR) overlapping with DMRs were selected for functional characterization. The promoters of genes were computed 2.0 kb upstream of the gene start site.