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Assay development

Priyanka A Shah, Pranav S Shrivastav, Jaivik V Shah, Archana George
A single LC-MS/MS assay is developed and validated for simultaneous determination of metformin and dapagliflozin in human plasma using ion-pair solid phase extraction. Chromatographic separation of the analytes and their internal standards was carried out on a reversed-phase ACE 5CN (150 × 4.6 mm, 5 μm) column using acetonitrile-15 mM ammonium acetate, pH 4.5 (70:30, v/v) as the mobile phase. To achieve higher sensitivity and selectivity for the analytes, mass spectrometric analysis was performed using polarity switching approach...
December 5, 2018: Biomedical Chromatography: BMC
Dongyang Wang, Jianjun Zhang, Zengjun Li, Jianjun Han, Yongsheng Gao, Ming Chen, Yanqing Li
Fibroblast growth factor 19 (FGF19) promotes tumor growth in various types of cancer, but its function has not been investigated in the context of colorectal adenoma. Here, we report that FGF19 expression was greater in colorectal adenoma than in normal tissues, as measured by an enzyme-linked immunosorbent assay, quantitative reverse-transcription PCR and immunohistochemistry. FGF19 expression was also elevated in a subset of human colon cancer cell lines. Moreover, FGF receptor 4 (FGFR4), the cognate receptor for FGF19, was upregulated in colorectal adenoma tissues...
December 5, 2018: Digestive Diseases
Junpu Li, Shaoshen Li, Lunhui Huang, Yaqiong Cui, Tiantian She, Ying Bian, Huiqiang Li
BACKGROUND: An increase in allergen-specific IgG4 (sIgG4 ), which serves as a blocking antibody, is associated with acquisition of immune tolerance after immunotherapy. In this study, we developed a rapid, sensitive, and homogeneous immunoassay based on the light-initiated chemiluminescent assay (LICA) technology for quantifying allergen sIgG4 in serum samples. METHODS: Allergen sIgG4 was measured in vitro by incubating the sample with biotinylated allergens and chemiluminescent beads coated with anti-human IgG4 antibody, followed by the addition of streptavidin-coated sensitizer beads...
December 2, 2018: Clinica Chimica Acta; International Journal of Clinical Chemistry
Fábio R M Leite, Christian Enevold, Klaus Bendtzen, Vibeke Baelum, Rodrigo López
BACKGROUND: Even though bacteria trigger inflammation, most of the tissue destruction in periodontitis is due to the host inflammatory response. In addition to immunological events that drive development of early periodontitis, numerous environmental factors like genetics and smoking play a role. We investigated whether the carriage of selected single nucleotide polymorphisms (SNP) of toll-like receptors (TLR), NOD-like receptors (NLR) and RIG-I-like receptors (RLR) was associated with the diagnosis of early periodontitis in a case-control study...
December 5, 2018: Journal of Periodontology
Marius Gheorghe, Geir Kjetil Sandve, Aziz Khan, Jeanne Chèneby, Benoit Ballester, Anthony Mathelier
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is the most popular assay to identify genomic regions, called ChIP-seq peaks, that are bound in vivo by transcription factors (TFs). These regions are derived from direct TF-DNA interactions, indirect binding of the TF to the DNA (through a co-binding partner), nonspecific binding to the DNA, and noise/bias/artifacts. Delineating the bona fide direct TF-DNA interactions within the ChIP-seq peaks remains challenging. We developed a dedicated software, ChIP-eat, that combines computational TF binding models and ChIP-seq peaks to automatically predict direct TF-DNA interactions...
December 4, 2018: Nucleic Acids Research
Nora Pisanic, Sarah-Blythe Ballard, Fabiola D Colquechagua, Ruthly François, Natalie Exum, Pablo Peñataro Yori, Kellogg J Schwab, Douglas A Granger, Barbara Detrick, Maribel Paredes Olortegui, Holger Mayta, Gerardo J Sánchez, Robert H Gilman, Christopher D Heaney, Jan Vinjé, Margaret N Kosek
Background: Norovirus is a leading cause of acute gastroenteritis worldwide. Routine norovirus diagnosis requires stool collection. The goal of this study was to develop and validate a noninvasive method to diagnose norovirus to complement stool diagnostics and to facilitate studies on transmission. Methods: A multiplex immunoassay to measure salivary immunoglobulin G (IgG) responses to 5 common norovirus genotypes (GI.1, GII.2, GII.4, GII.6, and GII.17) was developed...
December 5, 2018: Journal of Infectious Diseases
Sijie Fang, Yazhuo Huang, Ningjian Wang, Shuo Zhang, Sisi Zhong, Yinwei Li, Jing Sun, Xingtong Liu, Yang Wang, Ping Gu, Bin Li, Huifang Zhou, Xianqun Fan
Context: Unique features of local immunity in thyroid-associated ophthalmopathy (TAO) may affect disease progression. Objective: To investigate the association between the orbital immune microenvironment and TAO development. Design/Setting/Participants: TAO and control orbital connective tissues were collected. Main Outcome Measures: Single-cell sequencing examined orbital lymphocytic infiltrates. Multi-color flow cytometry explored the phenotypes of different cell subsets and in vitro models for cell functional studies...
December 3, 2018: Journal of Clinical Endocrinology and Metabolism
Beibei Wang, Haoyu Wang, Peiquan Li, Liangliang Wang, Hongli Liu, Jingbo Liu, Lihua Wang
OBJECTIVE: This study investigated serum interleukin-10 (IL-10) levels, changes in peripheral blood CD4+CD25+ regulatory T cell (PBCDT) ratios, and the prognosis of cervical cancer (CC) patients. METHODS: Seventy patients with CC composed the observation group, and 70 healthy subjects composed the control group. The PBCDT ratios in the CC patients and healthy subjects were calculated. Serum IL-10 levels were detected with a double antibody sandwich enzyme-linked immunosorbent assay (ELISA)...
December 3, 2018: Clinics
Kanny Diallo, Mamadou D Coulibaly, Lisa S Rebbetts, Odile B Harrison, Jay Lucidarme, Kadidja Gamougam, Yenenesh K Tekletsion, Akalifa Bugri, Aliou Toure, Bassira Issaka, Marietou Dieng, Caroline Trotter, Jean-Marc Collard, Samba O Sow, Xin Wang, Leonard W Mayer, Ray Borrow, Brian M Greenwood, Martin C J Maiden, Olivier Manigart
Improved methods for the detection and characterization of carried Neisseria meningitidis isolates are needed. We evaluated a multiplex PCR algorithm for the detection of a variety of carriage strains in the meningitis belt. To further improve the sensitivity and specificity of the existing PCR assays, primers for gel-based PCR assays (sodC, H, Z) and primers/probe for real-time quantitative PCR (qPCR) assays (porA, cnl, sodC, H, E, Z) were modified or created using Primer Express software. Optimized multiplex PCR assays were tested on 247 well-characterised carriage isolates from six countries of the African meningitis belt...
2018: PloS One
Ajay Ravindranathan, Beth Cimini, Morgan E Diolaiti, Bradley A Stohr
The telomerase enzyme enables unlimited proliferation of most human cancer cells by elongating telomeres and preventing replicative senescence. Despite the critical importance of telomerase in cancer biology, challenges detecting telomerase activity and expression in individual cells have hindered the ability to study patterns of telomerase expression and function across heterogeneous cell populations. While sensitive assays to ascertain telomerase expression and function exist, these approaches have proven difficult to implement at the single cell level...
2018: PloS One
Taiwo Samson Awolola, Adedapo Adeogun, Abiodun K Olakiigbe, Tolulope Oyeniyi, Yetunde Adeola Olukosi, Hilary Okoh, Tolulope Arowolo, Joel Akila, Adedayo Oduola, Chioma N Amajoh
Anopheles gambiae, An. coluzzii and An. arabiensis are the three major vectors of malaria in Nigeria. These mosquitoes have developed resistance to different insecticides. Insecticides resistance intensity assay was recently introduced to provide insight into the potential operational significance of insecticide resistance. Here, we present data on pyrethroids resistance intensity and resistance mechanisms from six vector surveillance sites (Lagos, Ogun, Edo, Anambra, Kwara and Niger) in Nigeria. Adult Anopheles reared from larval collections were tested using WHO insecticides susceptibility protocol with 1x concentration of permethrin and deltamethrin followed with intensity assays with 5x and 10x concentrations of both insecticides...
2018: PloS One
Min Zeng, Yulin Luo, Chunrong Xu, Rong Li, Ni Chen, Xin Deng, Dan Fang, Liqun Wang, Jianbo Wu, Mao Luo
Platelets (PLTs) - endothelial cells (ECs) interaction appears to contribute to phenotypic transition of VSMCs that play an important role in the physiological and pathological process of vascular complications in Type 2 diabetes (DM2). However, the precise mechanisms by which interactions between PLTs and ECs affect VSMCs phenotype, have largely remained unclear. We determined the effect of diabetic PLTs-ECs interaction to influence VSMCs migration, proliferation and phenotypic transformation in triple cell co-culture models using the qRT-PCR, western blot, fluorescence microscopy, wound scratch assays, CCK-8 assays, and gelatine zymography assays...
December 5, 2018: American Journal of Physiology. Cell Physiology
Jianjiao Wang, Feimin Chen, Yunhao Liu, Yuxuan Liu, Kunlong Li, Xiliang Yang, Shuwen Liu, Xuefeng Zhou, Jie Yang
A new spirocyclic γ-lactam, named spirostaphylotrichin X (1), and three related known spirostaphylotrichins (2-4) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures were determined by spectroscopic analyses. Spirostaphylotrichin X (1) displayed obvious inhibitory activities against multiple influenza virus strains, with IC50 values from 1.2 to 5.5 μM. Investigation of the mechanism showed that 1 inhibited viral polymerase activity and interfered with the production of progeny viral RNA...
December 5, 2018: Journal of Natural Products
Stefan Harth, Andre Ten Haaf, Christian Loew, Christian Frisch, Achim Knappik
Anti-idiotypic antibodies play an important role in pre-clinical and clinical development of therapeutic antibodies, where they are used for pharmacokinetic studies and for the development of immunogenicity assays. By using an antibody phage display library in combination with guided in vitro selection against various marketed drugs, we generated antibodies that recognize the drug only when bound to its target. We have named such specificities Type 3, to distinguish them from the anti-idiotypic antibodies that specifically detect free antibody drug or total drug...
December 5, 2018: MAbs
Josephine A Bonventre, Chelsea Holman, Aayushi Manchanda, Sara J Codding, Trisha Chau, Jacob Huegel, Carrie Barton, Robert Tanguay, Colin P Johnson
The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance however, many developmental genes have yet to be identified. We have determined that Fer1L6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1L6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1L6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity...
December 5, 2018: Molecular Biology of the Cell
Lisanne F van Dessel, Silvia R Vitale, Jean C A Helmijr, Saskia M Wilting, Michelle van der Vlugt-Daane, Esther Oomen-de Hoop, Stefan Sleijfer, John W M Martens, Maurice P H M Jansen, Martijn P Lolkema
The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high-throughput method for fast, reproducible and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study is to compare two competing automated DNA isolation platforms (Maxwell (MX) and QIAsymphony (QS)) to the current 'gold standard' QA to facilitate high-throughput processing of samples in prospective trials...
December 5, 2018: Molecular Oncology
Michela Zuffo, Xiao Xie, Anton Granzhan
High-throughput assessment of secondary structures adopted by DNA oligonucleotides is currently hampered by the lack of suitable biophysical methods. Fluorescent sensors hold great potential in this respect; however, the moderate selectivity that they display for one DNA conformation over the others constitutes a major drawback to the development of accurate assays. Moreover, the use of single sensors impedes a comprehensive classification of the tested sequences. Herein, we propose to overcome these limitations through the development of a fluorescence sensor array constituted by easily accessible, commercial dyes...
December 5, 2018: Chemistry: a European Journal
Xiaoxiao Jiang, Yanhong Cheng, Chaojie Hu, Aimei Zhang, Yingli Ren, Xiucai Xu
MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this study, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to imatinib-sensitive CML cells and patients with optimal responses respectively...
December 5, 2018: Leukemia & Lymphoma
Elaine Hui-Chien Lee, Sherlyn Sheau-Chin Lim, Kah-Hay Yuen, Chong-Yew Lee
OBJECTIVES: This study aims to investigate the blood-brain barrier (BBB) permeability of curcumin analogues with shortened linkers and their ability to protect against amyloid-beta toxicity in a whole organism model. METHOD: Four curcumin analogues were synthesized. These analogues and curcumin were evaluated for their BBB permeability in the parallel artificial membrane permeability assay. The transgenic Caenorhabditis elegansGMC101 that expresses human Aβ1-42 was treated with the compounds to evaluate their ability to delay Aβ-induced paralysis...
December 4, 2018: Journal of Pharmacy and Pharmacology
Fujin Shen, Hongyun Zheng, Limei Zhou, Wei Li, Xuexian Xu
Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells...
December 4, 2018: Journal of Cellular Physiology
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