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https://www.readbyqxmd.com/read/29772016/development-of-a-duplex-droplet-digital-pcr-assay-for-absolute-quantitative-detection-of-candidatus-liberibacter-asiaticus
#1
Vijayanandraj Selvaraj, Yogita Maheshwari, Subhas Hajeri, Jianchi Chen, Thomas Greg McCollum, Raymond Yokomi
Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors...
2018: PloS One
https://www.readbyqxmd.com/read/29769145/phylogenetic-relationships-stage-specific-expression-and-localisation-of-a-unique-family-of-inactive-cysteine-proteases-in-sarcoptes-scabiei
#2
Deepani D Fernando, Simone L Reynolds, Martha Zakrzewski, Ehtesham Mofiz, Anthony T Papenfuss, Deborah Holt, Katja Fischer
BACKGROUND: Scabies is worldwide one of the most common, yet neglected, parasitic skin infections, affecting a wide range of mammals including humans. Limited treatment options and evidence of emerging mite resistance against the currently used drugs drive our research to explore new therapeutic candidates. Previously, we discovered a multicopy family of genes encoding cysteine proteases with their catalytic sites inactivated by mutation (SMIPP-Cs). This protein family is unique in parasitic scabies mites and is absent in related non-burrowing mites...
May 16, 2018: Parasites & Vectors
https://www.readbyqxmd.com/read/29769099/circulating-esr1-mutations-at-the-end-of-aromatase-inhibitor-adjuvant-treatment-and-after-relapse-in-breast-cancer-patients
#3
Violette Allouchery, Ludivine Beaussire, Anne Perdrix, David Sefrioui, Laetitia Augusto, Cécile Guillemet, Nasrin Sarafan-Vasseur, Frédéric Di Fiore, Florian Clatot
BACKGROUND: Detection of circulating ESR1 mutations is associated with acquired resistance to aromatase inhibitor (AI) in metastatic breast cancer. Until now, the presence of circulating ESR1 mutations at the end of adjuvant treatment by AI in early breast cancer had never been clearly established. In this context, the aim of the present study was to evaluate the circulating ESR1 mutation frequency at the end of adjuvant treatment and after relapse. METHODS: This monocentric retrospective study was based on available stored plasmas and included all early breast cancer patients who completed at least 2 years of AI adjuvant treatment and experienced a documented relapse after the end of their treatment...
May 16, 2018: Breast Cancer Research: BCR
https://www.readbyqxmd.com/read/29765524/cross-platform-comparison-for-the-detection-of-ras-mutations-in-cfdna-ddpcr-biorad-detection-assay-beaming-assay-and-ngs-strategy
#4
Jessica Garcia, Julien Forestier, Eric Dusserre, Anne-Sophie Wozny, Florence Geiguer, Patrick Merle, Claire Tissot, Carole Ferraro-Peyret, Frederick S Jones, Daniel L Edelstein, Valérie Cheynet, Claire Bardel, Gaelle Vilchez, Zhenyu Xu, Pierre Paul Bringuier, Marc Barritault, Karen Brengle-Pesce, Marielle Guillet, Marion Chauvenet, Brigitte Manship, Marie Brevet, Claire Rodriguez-Lafrasse, Valérie Hervieu, Sébastien Couraud, Thomas Walter, Léa Payen
CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed...
April 20, 2018: Oncotarget
https://www.readbyqxmd.com/read/29765171/risks-and-protective-factors-for-triple-negative-breast-cancer-with-a-focus-on-micronutrients-and-infections
#5
Dagmar Horakova, Katerina Bouchalova, Karel Cwiertka, Ladislav Stepanek, Jana Vlckova, Helena Kollarova
Triple negative breast cancer (TNBC) is an aggressive form of breast cancer (BC) with a poor prognosis. Second, patients cannot benefit from targeted therapy, except for those with BRCA1/2 mutations, for whom poly (ADP-ribose) polymerase (PARP) inhibition therapy using olaparib has recently been approved. As global priorities continue to be epidemiological analysis of BC risk factors and early diagnosis, this review focuses on the risks and protective factors associated with TNBC. A PubMed keyword search for new knowledge on the risks and protective factors for TNBC was carried out...
May 15, 2018: Biomedical Papers of the Medical Faculty of the University Palacký, Olomouc, Czechoslovakia
https://www.readbyqxmd.com/read/29760807/quality-control-of-next-generation-sequencing-based-in-vitro-diagnostic-test-for-onco-relevant-mutations-using-multiplex-reference-materials-in-plasma
#6
Donglai Liu, Haiwei Zhou, Dawei Shi, Shu Shen, Yabin Tian, Lin Wang, Jiatao Lou, Rong Cong, Juan Lu, Henghui Zhang, Meiru Zhao, Shida Zhu, Zhisheng Cao, Ruilin Jin, Yin Wang, Xiaoni Zhang, Guohua Yang, Youchun Wang, Chuntao Zhang
Background: Widespread clinical implementation of next-generation sequencing (NGS)-based cancer in vitro diagnostic tests (IVDs) highlighted the urgency to establish reference materials which could provide full control of the process from nucleic acid extraction to test report generation. The formalin-fixed, paraffin-embedded (FFPE) tissue and blood plasma containing circulating tumor deoxyribonucleic acid (ctDNA) were mostly used for clinically detecting onco-relevant mutations. Methods: We respectively developed multiplex FFPE and plasma reference materials covering three clinically onco-relevant mutations within the epidermal growth factor receptor ( EGFR ) gene at serial allelic frequencies...
2018: Journal of Cancer
https://www.readbyqxmd.com/read/29759193/highly-sensitive-detection-and-mutational-analysis-of-lung-cancer-circulating-tumor-cells-using-integrated-combined-immunomagnetic-beads-with-a-droplet-digital-pcr-chip
#7
Wanlei Gao, Ting Huang, Haojun Yuan, Jun Yang, Qinghui Jin, Chunping Jia, Guoxin Mao, Jianlong Zhao
Circulating tumor cells (CTCs) have become an important biomarker for liquid biopsy to monitor tumor progression and indicate response to therapies. Many epithelial cellular adhesion molecule (EpCAM) dependent CTC isolation methods have been developed, which have a limitation for low EpCAM expressed tumor cells. In an effort to overcome these drawbacks, we developed combined immunomagnetic beads (EpCAM, Mucin1 and epidermal growth factor receptor) to sensitively isolate CTCs for immunofluorescence analysis and genetic characterization...
August 1, 2018: Talanta
https://www.readbyqxmd.com/read/29758488/genetic-variants-in-dyf-7-validated-by-droplet-digital-pcr-are-not-drivers-for-ivermectin-resistance-in-haemonchus-contortus
#8
Safaa T Elmahalawy, Peter Halvarsson, Moa Skarin, Johan Höglund
Resistance to ivermectin (IVM) in the nematode Haemonchus contortus in small ruminants is an increasing problem throughout the world. Access to molecular diagnostics will allow early detection of IVM resistance, which in turn can limit the spread of resistant isolates. One candidate gene which has recently been suggested as a marker for IVM resistance is that for dye-filling protein (dyf-7). In this study, we critically investigated the suitability of A141G and G153T single nucleotide polymorphisms (SNP) of dyf-7 as a marker in larval cultures collected from sheep farms in Sweden, involving several isolates for which resistance status had been characterised by the faecal egg count reduction test (FECRT)...
April 26, 2018: International Journal for Parasitology, Drugs and Drug Resistance
https://www.readbyqxmd.com/read/29758481/kaempferol-alleviates-palmitic-acid-induced-lipid-stores-endoplasmic-reticulum-stress-and-pancreatic-%C3%AE-cell-dysfunction-through-ampk-mtor-mediated-lipophagy
#9
Ritu Varshney, Rajat Varshney, Rutusmita Mishra, Sumeet Gupta, Debabrata Sircar, Partha Roy
Kaempferol, a natural flavonoid, has the beneficial effects of preserving pancreatic β-cell mass and function, but its action on β-cell lipid metabolism still remains elusive. Recently, autophagy has been reported to play a major role in lipid metabolism in various cell types, but its role in pancreatic β-cell's lipid metabolism is rarely reported. Here, we investigated the role of kaempferol-induced autophagy in inhibition of lipid stores, ER stress and β-cell dysfunction in palmitic acid-challenged RIN-5F cells and isolated pancreatic islets...
March 2, 2018: Journal of Nutritional Biochemistry
https://www.readbyqxmd.com/read/29753511/acute-adrenal-cortex-injury-during-cardiopulmonary-bypass-in-a-canine-model
#10
Bo Mei, Song Yang, Yuan Yue, Jian Hou, Keke Wang, Guangxian Chen, Mengya Liang, Zhongkai Wu
OBJECTIVE: Cardiopulmonary bypass (CPB) might induce systemic inflammatory responses that cause acute injuries to multiple organs. However, no direct evidence exists to determine whether CPB leads to adrenal cortex injury or to describe its underlying mechanism. METHODS: Twelve healthy adult beagles were randomly assigned into control and CPB groups. After cannulation, mild hypothermia CPB was performed in the CPB group but not in the control group. The serum concentrations of various cytokines, cortisol, and aldosterone were assessed...
April 11, 2018: Journal of Thoracic and Cardiovascular Surgery
https://www.readbyqxmd.com/read/29749584/droplet-digital-pcr-detects-high-rate-of-tp53-r249s-mutants-in-cell-free-dna-of-middle-african-patients-with-hepatocellular-carcinoma
#11
Agnès Marchio, Marie Amougou Atsama, Aubin Béré, Narcisse-Patrice Komas, Dominique Noah Noah, Paul Jean Adrien Atangana, Serge-Magloire Camengo-Police, Richard Njouom, Claudine Bekondi, Pascal Pineau
Hepatocellular carcinoma (HCC) is still a major killing malignancy in sub-Saharan Africa. Lifelong intoxication with aflatoxin B1 is considered as one of the primary causes of this situation. The role of aflatoxin in HCC from a given population is commonly estimated through the prevalence of R249S mutation of TP53, a hallmark for previous exposure to the mycotoxin. However, the role of AFB1 is barely known in large part of Africa. We conducted a survey on circulating cell-free DNA from 149 patients with HCC and 213 control subjects with and without liver diseases from Cameroon and Central African Republic using droplet digital PCR technique...
May 10, 2018: Clinical and Experimental Medicine
https://www.readbyqxmd.com/read/29746866/cav-1-promotes-atherosclerosis-by-activating-jnk-associated-signaling
#12
Dong-Xia Wang, Yong-Quan Pan, Bing Liu, Li Dai
The objective of the study is to calculate the role and underlying the molecular mechanisms of caveolin-1 (Cav-1) in atherosclerosis (AS). Cav-1 was mainly expressed in the endothelial cells of atherosclerotic lesions in both human patients and apolipoprotein E deficient (ApoE-/- ) mice. Cav-1 deficiency (Cav-1-/- ) attenuated high-fat diet (HFD)-induced atherosclerotic lesions in ApoE-/- mice, supported by the reduced aortic plaques. Cav-1-/- reduced the macrophage content and decreased the release of inflammation-related cytokines or chemokine in serum or abdominal aortas, accompanied with the inactivation of inhibitor κB kinase κ (IKKβ)/p65/IκBα signaling pathway...
May 7, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29745261/streptozotocin-induced-diabetes-alters-transcription-of-multiple-genes-necessary-for-extracellular-matrix-remodeling-in-rat-patellar-tendon
#13
Shivam H Patel, Arman Sabbaghi, Chad C Carroll
OVERVIEW: Tendon collagen fibril degradation is commonly seen in tendons of diabetics, but the mechanisms responsible for these changes remain to be elucidated. We have demonstrated that streptozotocin (STZ)-induced diabetes increases tendon cell proliferation and collagen content. In the present study, we evaluated that impact of STZ-induced diabetes on mRNA transcripts involved with collagen fibril organization, extracellular matrix (ECM) remodeling, apoptosis, and proliferation. MATERIALS AND METHODS: Rats were divided into four groups: nondiabetic (control, n = 9), 1 week (acute, n = 8) or 10 weeks of diabetes (chronic, n = 7), and 10 weeks of diabetes with insulin (insulin, n = 8)...
May 10, 2018: Connective Tissue Research
https://www.readbyqxmd.com/read/29743667/evaluation-of-pre-analytical-factors-affecting-plasma-dna-analysis
#14
Havell Markus, Tania Contente-Cuomo, Maria Farooq, Winnie S Liang, Mitesh J Borad, Shivan Sivakumar, Simon Gollins, Nhan L Tran, Harshil D Dhruv, Michael E Berens, Alan Bryce, Aleksandar Sekulic, Antoni Ribas, Jeffrey M Trent, Patricia M LoRusso, Muhammed Murtaza
Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly...
May 9, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29740475/dose-response-and-prediction-characteristics-of-a-methylation-sensitive-digital-pcr-assay-for-cigarette-consumption-in-adults
#15
Robert Philibert, Meesha Dogan, Amanda Noel, Shelly Miller, Brianna Krukow, Emma Papworth, Joseph Cowley, Jeffrey D Long, Steven R H Beach, Donald W Black
The tobacco use disorders are the largest preventable cause of morbidity and mortality in the world. A substantial barrier to the development of better intervention and screening measures is the lack of clinically employable biomarkers to detect the existence and extent of tobacco consumption. In prior work, we and others have shown that array based assessment of DNA methylation status at cg05575921 is a sensitive and quantitative method for assessing cigarette consumption. Unfortunately, in general, arrays are not practical clinical tools...
2018: Frontiers in Genetics
https://www.readbyqxmd.com/read/29739972/cell-type-specific-expression-of-oncogenic-and-tumor-suppressive-micrornas-in-the-human-prostate-and-prostate-cancer
#16
Binod Kumar, Avi Z Rosenberg, Su Mi Choi, Karen Fox-Talbot, Angelo M De Marzo, Larisa Nonn, W Nathaniel Brennen, Luigi Marchionni, Marc K Halushka, Shawn E Lupold
MiR-1 and miR-143 are frequently reduced in human prostate cancer (PCa), while miR-141 and miR-21 are frequently elevated. Consequently, these miRNAs have been studied as cell-autonomous tumor suppressors and oncogenes. However, the cell-type specificity of these miRNAs is not well defined in prostate tissue. Through two different microdissection techniques, and droplet digital RT-PCR, we quantified these miRNAs in the stroma and epithelium of radical prostatectomy specimens. In contrast to their purported roles as cell-autonomous tumor suppressors, we found miR-1 and miR-143 expression to be predominantly stromal...
May 8, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29736807/quantitative-detection-of-low-abundance-transcripts-at-single-cell-level-in-human-epidermal-keratinocytes-by-digital-droplet-reverse-transcription-polymerase-chain-reaction
#17
Frédéric Auvré, Julien Coutier, Michèle T Martin, Nicolas O Fortunel
Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized...
May 8, 2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29734217/dysregulation-of-apoptosis-and-autophagy-gene-expression-in-pbmcs-of-efficiently-treated-hiv-infected-patients
#18
Amandine Serrano, Saïd El Haddad, Frédéric Moal, Thierry Prazuck, Eric Legac, Chloé Robin, Fabienne Brule, Stéphane Charpentier, Thierry Normand, Alain Legrand, Laurent Hocqueloux, Lucile Mollet
OBJECTIVE: We measure the transcript levels of the pro-apoptotic GALIG, anti-apoptotic MCL1 genes and those of the autophagy genes BECN1, MAP1LC3B, ATG9a, P62/SQSTM1, GABARAP, GABARAPL1 and GABARAPL2 to define if mRNA alteration can characterize HIV infected patients effectively treated with combined antiretroviral therapy (cART). DESIGN: Monocentric pilot study conducted on PBMC of 40 uninfected donors and 27 HIV positive patients effectively treated by cART for at least 8...
May 3, 2018: AIDS
https://www.readbyqxmd.com/read/29727699/pyrosequencing-based-quantitative-measurement-of-calr-mutation-allele-burdens-and-their-clinical-implications-in-patients-with-myeloproliferative-neoplasms
#19
Yejin Oh, Ik-Chan Song, Jimyung Kim, Gye Cheol Kwon, Sun Hoe Koo, Seon Young Kim
BACKGROUND: We developed a pyrosequencing-based method for the quantification of CALR mutations and compared the results using Sanger sequencing, fragment length analysis (FLA), digital-droplet PCR (ddPCR), and next-generation sequencing (NGS). METHODS: Method validation studies were performed using cloned plasmid controls. Samples from 24 patients with myeloproliferative neoplasms were evaluated. RESULTS: Among the 24 patients, 15 had CALR mutations (7 type 1, 2 type 2, and 6 other mutations)...
May 1, 2018: Clinica Chimica Acta; International Journal of Clinical Chemistry
https://www.readbyqxmd.com/read/29725728/validated-reverse-transcription-droplet-digital-pcr-serves-as-a-higher-order-method-for-absolute-quantification-of-potato-virus-y-strains
#20
Nataša Mehle, David Dobnik, Maja Ravnikar, Maruša Pompe Novak
RNA viruses have a great potential for high genetic variability and rapid evolution that is generated by mutation and recombination under selection pressure. This is also the case of Potato virus Y (PVY), which comprises a high diversity of different recombinant and non-recombinant strains. Consequently, it is hard to develop reverse transcription real-time quantitative PCR (RT-qPCR) with the same amplification efficiencies for all PVY strains which would enable their equilibrate quantification; this is specially needed in mixed infections and other studies of pathogenesis...
May 3, 2018: Analytical and Bioanalytical Chemistry
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