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Lifestyle modifications result in alterations in the gut microbiota in obese children.
Cho, KY
BMC microbiology. 2021;(1):10
Abstract
BACKGROUND The association between the gut microbiota and pediatric obesity was analyzed in a cross-sectional study. A prospective study of obese children was conducted to assess the gut microbial alterations after a weight change. We collected fecal samples from obese children before and after a 2-month weight reduction program that consisted of individual counseling for nutritional education and physical activity, and we performed 16S rRNA gene amplicon sequencing using an Illumina MiSeq platform. RESULTS Thirty-six participants, aged 7 to 18 years, were classified into the fat loss (n = 17) and the fat gain (n = 19) groups according to the change in total body fat (%) after the intervention. The baseline analysis of the gut microbiota in the preintervention stages showed dysbiotic features of both groups compared with those of normal-weight children. In the fat loss group, significantly decreased proportions of Bacteroidetes phylum, Bacteroidia class, Bacteroidales order, Bacteroidaceae family, and Bacteroides genus, along with increased proportions of Firmicutes phylum, Clostridia class, and Clostridiales order, were observed after intervention. The microbial richness was significantly reduced, without a change in beta diversity in the fat loss group. The fat gain group showed significantly deceased proportions of Firmicutes phylum, Clostridia class, Clostridiales order, Lachnospiraceae family, and Eubacterium hallii group genus, without a change in diversity after the intervention. According to the functional metabolic analysis by the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2, the "Nitrate Reduction VI" and "Aspartate Superpathway" pathways were predicted to increase significantly in the fat loss group. The cooccurring networks of genera were constructed and showed the different microbes that drove the changes between the pre- and postintervention stages in the fat loss and fat gain groups. CONCLUSIONS This study demonstrated that lifestyle modifications can impact the composition, richness, and predicted functional profiles of the gut microbiota in obese children after weight changes. TRIAL REGISTRATION ClinicalTrials.gov NCT03812497 , registration date January 23, 2019, retrospectively registered.
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Pretreatment Tumor DNA Sequencing of KIT and PDGFRA in Endosonography-Guided Biopsies Optimizes the Preoperative Management of Gastrointestinal Stromal Tumors.
Hedenström, P, Andersson, C, Sjövall, H, Enlund, F, Nilsson, O, Nilsson, B, Sadik, R
Molecular diagnosis & therapy. 2020;(2):201-214
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Abstract
BACKGROUND Neoadjuvant tyrosine kinase inhibitor (TKI) therapy increases the chance of organ-preserving, radical resection in selected patients with gastrointestinal stromal tumors (GISTs). We aimed to evaluate systematic, immediate DNA sequencing of KIT and PDGFRA in pretreatment GIST tissue to guide neoadjuvant TKI therapy and optimize preoperative tumor response. METHODS All patients who were candidates for neoadjuvant therapy of a suspected GIST [the study cohort (SC)] were prospectively included from January 2014 to March 2018. Patients were subjected to pretreatment endosonography-guided fine-needle biopsy (EUS-FNB) or transabdominal ultrasound-guided needle biopsy (TUS-NB), followed by immediate tumor DNA sequencing (< 2 weeks). A historic (2006-2013) reference cohort (RC) underwent work-up without sequencing before neoadjuvant imatinib (n = 42). The rate of optimal neoadjuvant therapy (TherapyOPTIMAL) was calculated, and the induced tumor size reduction (Tumor RegressionMAX, %) was evaluated by computed tomography (CT) scan. RESULTS The success rate of pretreatment tumor DNA sequencing in the SC (n = 81) was 77/81 (95%) [EUS-FNB 71/74 (96%); TUS-NB 6/7 (86%)], with mutations localized in KIT (n = 58), PDGFRA (n = 18), or neither gene, wild type (n = 5). In patients with a final indication for neoadjuvant therapy, the TherapyOPTIMAL was higher in the SC compared with the RC [61/63 (97%) versus 33/42 (79%), p = 0.006], leading to a significantly higher Tumor RegressionMAX in patients treated with TKI (27% vs. 19%, p = 0.015). CONCLUSIONS Pretreatment endosonography-guided biopsy sampling followed by immediate tumor DNA sequencing of KIT and PDGFRA is highly accurate and valuable in guiding neoadjuvant TKI therapy in GIST. This approach minimizes maltreatment with inappropriate regimens and leads to improved tumor size reduction before surgery.
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Comparison of methods for quantification of global DNA methylation in human cells and tissues.
Lisanti, S, Omar, WA, Tomaszewski, B, De Prins, S, Jacobs, G, Koppen, G, Mathers, JC, Langie, SA
PloS one. 2013;(11):e79044
Abstract
DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.
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Performance of Applied Biosystems ViroSeq HIV-1 Genotyping System for sequence-based analysis of non-subtype B human immunodeficiency virus type 1 from Uganda.
Mracna, M, Becker-Pergola, G, Dileanis, J, Guay, LA, Cunningham, S, Jackson, JB, Eshleman, SH
Journal of clinical microbiology. 2001;(12):4323-7
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Abstract
The Applied Biosystems ViroSeq HIV-1 Genotyping System is a commercially available, integrated system for sequence-based analysis of drug resistance mutations in human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for analysis of non-subtype B HIV-1 by analyzing plasma samples from Ugandan women and infants. Plasma samples were obtained from 105 women and 25 infants enrolled in a Ugandan clinical trial. HIV-1 analysis was performed with the ViroSeq system according to the manufacturer's instructions, except that the volume of plasma used for analysis was less than the recommended 0.5 ml for some samples. Viral loads ranged from 2,313 to 2,336,400 copies/ml. PCR products suitable for sequencing were amplified from all samples tested. Complete sequences for protease (amino acids 1 to 99) and RT (amino acids 1 to 320) were obtained for 102 of 105 (97%) of the maternal samples tested and all 25 of the infant samples tested. Complete double-stranded sequences were obtained for 90 of 105 (86%) of the maternal samples tested and 22 of 25 (88%) of the infant samples tested. The sequences obtained with this system were used for HIV-1 subtyping. The subtypes identified were A, C, D, and A/D recombinant HIV-1. The performances of the seven sequencing primers were similar for the subtypes examined. The ViroSeq system performs well for analysis of Ugandan plasma samples with subtypes A, C, D, and A/D recombinant HIV-1. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in countries where these subtypes are prevalent.
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Comparison of sequencing by hybridization and cycle sequencing for genotyping of human immunodeficiency virus type 1 reverse transcriptase.
Hanna, GJ, Johnson, VA, Kuritzkes, DR, Richman, DD, Martinez-Picado, J, Sutton, L, Hazelwood, JD, D'Aquila, RT
Journal of clinical microbiology. 2000;(7):2715-21
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The performances of two methods of nucleotide sequencing were compared for the detection of drug resistance mutations in human immunodeficiency virus type 1 reverse transcriptase (RT) in viruses isolated from highly RT inhibitor-experienced individuals. Of 11,677 amino acids deduced from population PCR products by both cycle sequencing and sequencing by hybridization to high-density arrays of oligonucleotide probes, 97.4% were concordant by both methods, 0.8% were discordant, and 1.7% had an ambiguous determination by at least one method. A higher rate of discordance (3.9%) was observed among RT inhibitor resistance-associated codons. In 45% of the isolates, RT codon 67 was deduced as the wild-type Asp by hybridization sequencing but as the zidovudine resistance-associated Asn by cycle sequencing. In other resistance-associated codon discordances, cycle sequencing also more commonly called a known resistance-associated amino acid than hybridization sequencing did. The nucleotide sequence in the vicinity of several codons with discordant calls influenced population-based hybridization sequencing. For isolates evaluated by additional sequencing of molecular clones of PCR products by both methods, the discordance between methods was less frequent (0.4% of all 5,994 amino acids and 0 of 494 drug resistance-associated codons). At positions which were discordant or ambiguous in the population sequences, the results of sequencing of clones by both methods were usually in agreement with the population cycle sequencing result. In summary, most RT codons were highly concordant by both methods of population-based sequencing, with discordances due in large part to genetic mixtures within or adjacent to discordant codons.