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https://www.readbyqxmd.com/read/28807002/high-resolution-measurement-of-duf1220-domain-copy-number-from-whole-genome-sequence-data
#1
David P Astling, Ilea E Heft, Kenneth L Jones, James M Sikela
BACKGROUND: DUF1220 protein domains found primarily in Neuroblastoma BreakPoint Family (NBPF) genes show the greatest human lineage-specific increase in copy number of any coding region in the genome. There are 302 haploid copies of DUF1220 in hg38 (~160 of which are human-specific) and the majority of these can be divided into 6 different subtypes (referred to as clades). Copy number changes of specific DUF1220 clades have been associated in a dose-dependent manner with brain size variation (both evolutionarily and within the human population), cognitive aptitude, autism severity, and schizophrenia severity...
August 14, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28802157/droplet-digital-pcr-for-rapid-enumeration-of-viral-genomes-and-particles-from-cells-and-animals-infected-with-orthopoxviruses
#2
Jeffrey L Americo, Patricia L Earl, Bernard Moss
Droplet digital polymerase chain reaction (ddPCR) was adapted for quantifying the number of orthopoxviral genomes in purified virus samples, infected cell lysates and tissues of infected animals. In contrast to the more commonly used qPCR, the newer ddPCR provides absolute numbers of DNA copies in samples without need for standard curves and has the ability to detect rare mutants in a population. The genome/infectious unit ratio for several sucrose gradient-purified orthopoxviruses varied from 5 to 10, which correlated well with values obtained using the Virocyt, a dedicated fluorescence flow cytometer...
August 9, 2017: Virology
https://www.readbyqxmd.com/read/28796824/droplet-digital-polymerase-chain-reaction-ddpcr-assays-integrated-with-an-internal-control-for-quantification-of-bovine-porcine-chicken-and-turkey-species-in-food-and-feed
#3
Hanan R Shehata, Jiping Li, Shu Chen, Helen Redda, Shumei Cheng, Nicole Tabujara, Honghong Li, Keith Warriner, Robert Hanner
Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures...
2017: PloS One
https://www.readbyqxmd.com/read/28780426/characterization-of-oseltamivir-resistant-influenza-virus-populations-in-immunosuppressed-patients-using-digital-droplet-pcr-comparison-with-qpcr-and-next-generation-sequencing-analysis
#4
Maxime Pichon, Alexandre Gaymard, Laurence Josset, Martine Valette, Gilles Millat, Bruno Lina, Vanessa Escuret
INTRODUCTION: The H275Y substitution in neuraminidase (NA) confers oseltamivir-resistance in A(H1N1) influenza viruses (IV). Droplet digital PCR (ddPCR) is a new technique to explore single nucleotide polymorphisms. The aim of this study was to compare the performances of reverse transcriptase (RT)-ddPCR, RT-qPCR and next generation sequencing (NGS). We also analyzed the proportions of H275Y-NA substitution for two immunosuppressed patients with sustained shedding of A(H1N1)pdm09 IV. METHODS: RT-qPCR was performed using the ABI7500 platform...
August 3, 2017: Antiviral Research
https://www.readbyqxmd.com/read/28771147/comparison-of-droplet-digital-pcr-and-quantitative-pcr-for-the-detection-of-salmonella-and-its-application-for-river-sediments
#5
Gulshan Singh, Ayanda Sithebe, Abimbola M Enitan, Sheena Kumari, Faizal Bux, Thor Axel Stenström
Despite advances in microbial detection that quantitative polymerase chain reaction (qPCR) has led to, complex environmental samples, such as sediments, remain a challenge due to presence of PCR inhibitors. Aquatic sediments accumulate particle-bound microbial contaminants and thereby reflect a cumulative microbial load over time. The relatively new droplet digital PCR (ddPCR) has emerged as a direct quantitative method, highly tolerant to PCR inhibitors and relinquishing the necessity for calibration/standard curves...
August 2017: Journal of Water and Health
https://www.readbyqxmd.com/read/28754961/the-maintenance-of-telomere-length-in-cd28-t-cells-during-t-lymphocyte-stimulation
#6
Ejun Elijah Huang, Enzo Tedone, Ryan O'Hara, Crystal Cornelius, Tsung-Po Lai, Andrew Ludlow, Woodring E Wright, Jerry W Shay
Telomerase activity is not readily detected in resting human T lymphocytes, however upon antigen presentation, telomerase is transiently upregulated. Presently, it is not known if telomerase activation is necessary for the proliferation of T cells or for the maintenance of telomere lengths. In this study, we found that telomerase activation is not required for the short- term proliferation of T cells and that telomeres progressively shorten in a heterogeneous population of T cells, even if telomerase is detected...
July 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28754904/robust-and-accurate-digital-measurement-for-her2-amplification-in-her2-equivocal-breast-cancer-diagnosis
#7
Yuefeng Wang, Julia Y S Tsang, Yongmei Cui, Ji Cui, Ying Lin, Songli Zhao, Patrick T W Law, Sai Yin Cheung, Enders K O Ng, Gary M K Tse, Zunfu Ke
Currently, there are no recommended alternative assays for HER2 cases deemed equivocal by immunohistochemistry and fluorescent in situ hybridization. Digital PCR (ddPCR), a highly accurate method to determine DNA copy number, could be a robust alternative for clinical HER2 diagnostics. HER2 and CEP17 copy numbers were quantified using two ddPCR platforms (QX200 and RainDrop) in 102 samples of invasive breast cancers. Compared to routine assays, ddPCR gave a sensitivity and specificity of 82.8% and 97.3% respectively, with a kappa value of 0...
July 28, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28748500/digital-droplet-pcr-ddpcr-for-the-detection-and-quantification-of-hpv-16-18-33-and-45%C3%A2-a-short-report
#8
Gabriella Lillsunde Larsson, Gisela Helenius
PURPOSE: Human papilloma virus (HPV) infection is associated with several anogenital malignancies. Here, we set out to evaluate digital droplet PCR (ddPCR) as a tool for HPV 16, 18, 33 and 45 viral load quantification and, in addition, to compare the efficacy of the ddPCR assay for HPV 16 detection with that of quantitative real-time PCR (qPCR). METHODS: Clinical samples, positive for HPV genotypes 16, 18, 33 and 45 were analyzed for viral load using ddPCR. Sample DNA was cleaved before droplet generation and PCR...
July 26, 2017: Cellular Oncology (Dordrecht)
https://www.readbyqxmd.com/read/28743297/identification-of-reference-mirnas-in-plasma-useful-for-the-study-of-oestrogen-responsive-mirnas-associated-with-acquired-protein-s-deficiency-in-pregnancy
#9
J W Tay, I James, Q W Hughes, J Y Tiao, R I Baker
BACKGROUND: Accumulating evidence indicate that circulating microRNAs (miRNAs) are useful independent non-invasive biomarkers, with unique miRNA signatures defined for various pathophysiological conditions. However, there are no established universal housekeeping miRNAs for the normalisation of miRNAs in body fluids. We have previously identified an oestrogen-responsive miRNA, miR-494, in regulating the anticoagulant, Protein S, in HuH-7 liver cells. Moreover, increased thrombotic risk associated with elevated circulating oestrogen levels is frequently observed in pregnant women and oral contraceptive users...
July 25, 2017: BMC Research Notes
https://www.readbyqxmd.com/read/28737741/rapidly-inducible-cas9-and-dsb-ddpcr-to-probe-editing-kinetics
#10
John C Rose, Jason J Stephany, William J Valente, Bridget M Trevillian, Ha V Dang, Jason H Bielas, Dustin J Maly, Douglas M Fowler
We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL-BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs...
July 24, 2017: Nature Methods
https://www.readbyqxmd.com/read/28733559/dynamics-of-egfr-mutations-in-plasma-recapitulates-the-clinical-response-to-egfr-tkis-in-nsclc-patients
#11
Liwen Xiong, Shaohua Cui, Jingyan Ding, Yun Sun, Longfu Zhang, Yizhuo Zhao, Aiqin Gu, Tianqing Chu, Huimin Wang, Hua Zhong, Xin Ye, Yi Gu, Xin Zhang, Min Hu, Liyan Jiang
OBJECTIVES: Genomic profiling using plasma cell-free DNA (cfDNA) represents a non-invasive alternative to tumor re-biopsy, which is challenging in clinical practice. The feasibility of dynamically monitoring epidermal growth factor receptor (EGFR) mutation status using serial plasma samples from non-small cell lung cancer (NSCLC) patients treated by tyrosine kinase inhibitors (TKIs) and its application in tracking clinical response and detection of resistance were investigated. PATIENTS AND METHODS: Forty-five NSCLC patients with EGFR mutation-positive pre-TKI plasma and at least two post-TKI plasma collections were recruited to this study...
July 10, 2017: Oncotarget
https://www.readbyqxmd.com/read/28725028/detection-and-quantitation-of-copy-number-variation-in-the-voltage-gated-sodium-channel-gene-of-the-mosquito-culex-quinquefasciatus
#12
Walter Fabricio Silva Martins, Krishanthi Subramaniam, Keith Steen, Henry Mawejje, Triantafillos Liloglou, Martin James Donnelly, Craig Stephen Wilding
Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides...
July 19, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28724560/accurate-detection-of-methicillin-resistant-staphylococcus-aureus-in-mixtures-utilizing-single-bacterial-duplex-droplet-digital-pcr
#13
Jun Luo, Junhua Li, Hang Yang, Junping Yu, Hongping Wei
Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from the co-existence of methicillin-susceptible S. aureus (MSSA) and other methicillin-resistant Staphylococci In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swab. The new molecular assay is based on detecting the co-occurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with a chimeric lysin ClyH for cell lysis...
July 19, 2017: Journal of Clinical Microbiology
https://www.readbyqxmd.com/read/28722675/using-droplet-digital-pcr-to-analyze-mycn-and-alk-copy-number-in-plasma-from-patients-with-neuroblastoma
#14
Marco Lodrini, Annika Sprüssel, Kathy Astrahantseff, Daniela Tiburtius, Robert Konschak, Holger N Lode, Matthias Fischer, Ulrich Keilholz, Angelika Eggert, Hedwig E Deubzer
The invasive nature of surgical biopsies deters sequential application, and single biopsies often fail to reflect tumor dynamics, intratumor heterogeneity and drug sensitivities likely to change during tumor evolution and treatment. Implementing molecular characterization of cell-free neuroblastoma-derived DNA isolated from blood plasma could improve disease assessment for treatment selection and monitoring of patients with high-risk neuroblastoma. We established droplet digital PCR (ddPCR) protocols for MYCN and ALK copy number status in plasma from neuroblastoma patients...
July 7, 2017: Oncotarget
https://www.readbyqxmd.com/read/28720421/assessment-of-the-hiv-1-reservoir-in-cd4-regulatory-t-cells-by-a-droplet-digital-pcr-based-approach
#15
Gábor Artúr Dunay, Anastasia Solomatina, Silke Kummer, Anja Hüfner, Julia Katharina Bialek, Johanna Maria Eberhard, Eva Tolosa, Joachim Hauber, Julian Schulze Zur Wiesch
The relative contribution of regulatory T cells (Treg) as reservoir of HIV-1 in patients on chronic antiretroviral therapy is unclear to date. The aim of the current study was to assess the total HIV DNA burden and replication competent viral reservoir in Treg in comparison to central and effector memory cells (Tcm and Tem). Peripheral blood mononuclear cells were obtained from 10 HIV patients treated with antiretroviral therapy and one HIV elite controller. Droplet Digital PCR (ddPCR) was used to quantify total HIV DNA loads in FACS sorted CD4+ Treg (CD25+CD127lo) as compared with Tcm (CD45RO+CCR7+) and Tem (CD45RO+CCR7-)...
July 15, 2017: Virus Research
https://www.readbyqxmd.com/read/28714107/the-braf-and-nras-mutation-prevalence-in-dermoscopic-subtypes-of-acquired-naevi-reveals-constitutive-mapk-pathway-activation
#16
J M Tan, L N Tom, K Jagirdar, D Lambie, H Schaider, R A Sturm, H P Soyer, M S Stark
BACKGROUND: Acquired naevi can have unique dermoscopic patterns that correspond to distinct microanatomical growth patterns. Previous studies on acquired naevi stratified according to dermoscopic pattern, focused on the frequency of somatic BRAF mutations, whereas NRAS mutations remained to be elucidated. OBJECTIVES: To investigate the BRAF and NRAS mutation prevalence and activation of the MAPK pathway in distinct dermoscopic subtypes of acquired naevi. METHODS: Common mutations present in BRAF and NRAS were assessed in 40 globular, reticular, and peripheral rim of globules (PG) subtypes of acquired naevi from 27 participants (19 male, 8 female; mean age 46...
July 17, 2017: British Journal of Dermatology
https://www.readbyqxmd.com/read/28711990/assessment-of-braf-v600e-mutation-in-pulmonary-langerhans-cell-histiocytosis-in-tissue-biopsies-and-bronchoalveolar-lavages-by-droplet-digital-polymerase-chain-reaction
#17
Clémence Pierry, Charline Caumont, Elodie Blanchard, Camille Brochet, Gael Dournes, Audrey Gros, Thomas Bandres, Séverine Verdon, Marion Marty, Hugues Bégueret, Jean-Philippe Merlio
The neoplastic nature of pulmonary Langerhans cell histiocytosis (PLCH) is still debated. As the detection of BRAF (V600E) and MAP2K1 mutations in patients with PCLH is now considered for such assessment, the aim of our study was to evaluate digital droplet polymerase chain reaction (ddPCR) in PCLH diagnosis. We retrospectively analyzed BRAF(V600E) detection in a cohort of 42 PCLH tissues and 18 bronchoalveolar lavages (BALs) by ddPCR, immunohistochemistry, high-resolution melting PCR (HRM), and next-generation sequencing (NGS)...
July 15, 2017: Virchows Archiv: An International Journal of Pathology
https://www.readbyqxmd.com/read/28711173/presymptomatic-diagnosis-of-spinal-muscular-atrophy-through-newborn-screening
#18
Yin-Hsiu Chien, Shu-Chuan Chiang, Wen-Chin Weng, Ni-Chung Lee, Ching-Jie Lin, Wu-Shiun Hsieh, Wang-Tso Lee, Yuh-Jyh Jong, Tsang-Ming Ko, Wuh-Liang Hwu
OBJECTIVE: To demonstrate the feasibility of presymptomatic diagnosis of spinal muscular atrophy (SMA) through newborn screening (NBS). STUDY DESIGN: We performed a screening trial to assess all newborns who underwent routine newborn metabolic screening at the National Taiwan University Hospital newborn screening center between November 2014 and September 2016. A real-time polymerase chain reaction (RT-PCR) genotyping assay for the SMN1/SMN2 intron 7 c.888+100A/G polymorphism was performed to detect homozygous SMN1 deletion using dried blood spot (DBS) samples...
July 12, 2017: Journal of Pediatrics
https://www.readbyqxmd.com/read/28694163/mice-maintain-predominantly-maternal-g%C3%AE-s-expression-throughout-life-in-brown-fat-tissue-bat-but-not-other-tissues
#19
Olta Tafaj, Steven Hann, Ugur Ayturk, Matthew L Warman, Harald Jüppner
The murine Gnas (human GNAS) locus gives rise to Gαs and different splice variants thereof. The Gαs promoter is not methylated thus allowing biallelic expression in most tissues. In contrast, the alternative first Gnas/GNAS exons and their promoters undergo parent specific methylation, which limits transcription to the non-methylated allele. Pseudohypoparathyroidism type Ia (PHP1A) or type Ib (PHP1B) are caused by heterozygous maternal GNAS mutations suggesting that little or no Gαs is derived in some tissues from the non-mutated paternal GNAS thereby causing hormonal resistance...
July 8, 2017: Bone
https://www.readbyqxmd.com/read/28692472/quantification-of-immune-dysregulation-by-next-generation-polymerase-chain-reaction-to-improve-sepsis-diagnosis-in-surgical-patients
#20
Raquel Almansa, Alicia Ortega, Ana Ávila-Alonso, Maria Heredia-Rodríguez, Silvia Martín, Diana Benavides, Marta Martín-Fernandez, Lucia Rico, César Aldecoa, Jesús Rico, Iñigo López de Cenarruzabeitia, Juan Beltrán de Heredia, Esther Gomez-Sanchez, Marta Aragón, Cristina Andrés, Dolores Calvo, David Andaluz-Ojeda, Pilar Liu, Francisco Blanco-Antona, Lydia Blanco, Jose Ignacio Gómez-Herreras, Eduardo Tamayo, Jesus F Bermejo-Martin
OBJECTIVES: To quantify immunological dysfunction in surgical patients with presence/absence of sepsis using a droplet digital polymerase chain reaction (ddPCR) transcriptomic analysis. The study also aims to evaluate this approach for improving identification of sepsis in these patients. BACKGROUND: Immune dysregulation is a central event in sepsis. Quantification of the expression of immunological genes participating in the pathogenesis of sepsis could represent a new avenue to improve its diagnosis...
July 7, 2017: Annals of Surgery
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