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Assay development

Krishnadas Madhu, Prakash T
BACKGROUND: During the development of Multiple Sclerosis (MS) there is a marked activation of microglia and astrocyte, leading to progressive inflammation and degeneration of myelin sheath which results in axonal loss and neuron damage. PURPOSE: In this study, we have explored the action of Asiaticoside A against the activated microglia and astrocytes. METHODS: Primary microglia and astrocyte cultures were used for the study and the activity were evaluated using cell proliferation assay, nitrate assay and TNFα estimation using ELISA...
August 8, 2018: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
Butian Zhang, Lili Yu, Ning Han, Zhenzhen Hu, Shuang Wang, Lei Ding, Jinlan Jiang
OBJECTIVE: The purpose of this study was to find out the important lncRNA-miRNA-mRNA axis and pathway in osteosarcoma (OS) through bioinformatics analysis and verify the biological functions of lncRNA/miRNA/mRNA in OS through in vitro and in vivo assays. METHODS: The differential expression mRNAs and lncRNAs were identified through microarray analysis, and the altered pathways were identified by GSEA. The Pearson Coefficient was used to analyze the correlations between mRNAs and lncRNAs...
August 8, 2018: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
Wan-Ming Li, Lin-Lin Zhou, Min Zheng, Jin Fang
Circulating tumor cells (CTCs) have the potential to predict metastasis, and the capture of CTCs based on their surface markers is mostly applied for CTC detection. Considering that the CTCs with a metastatic phenotype preferably form a metastatic focus and that aptamers have the ability to bind targets with high specificity and affinity, we selected aptamers directed toward metastatic cells by subtractive Cell-SELEX technology using highly metastatic MDA-MB-231 cells as the target cell and low-metastatic MCF-7 cells as the negative cell for the capture of metastatic CTCs...
July 19, 2018: Molecular Therapy. Nucleic Acids
Yanli Lin, Qixin Leng, Min Zhan, Feng Jiang
The early detection of lung cancer is a major clinical challenge. Long noncoding RNAs (lncRNAs) have important functions in tumorigenesis. Plasma lncRNAs directly released from primary tumors or the circulating cancer cells might provide cell-free cancer biomarkers. The objective of this study was to investigate whether the lncRNAs could be used as plasma biomarkers for early-stage lung cancer. By using droplet digital polymerase chain reaction, we determined the diagnostic performance of 26 lung cancer-associated lncRNAs in plasma of a development cohort of 63 lung cancer patients and 33 cancer-free individuals, and a validation cohort of 39 lung cancer patients and 28 controls...
August 8, 2018: Translational Oncology
Tobias Maetzig, Michael Morgan, Axel Schambach
Hematopoiesis depends on the controlled differentiation of hematopoietic stem cells to mature cells with defined functions. Although each cell population within the hematopoietic hierarchy can be described by phenotypic markers, isolation of marker pure populations does not necessarily result in cells with homogeneous functionality. However, techniques that enable the efficient characterization of cell behavior with high resolution are limited. While single cell transplantation assays demand high mouse numbers and workload, sequencing-based fate tracking techniques require the destruction of the host cell, substantial financial resources, bioinformatics expertise, and suffer from a delay between sample acquisition and data interpretation...
August 8, 2018: Experimental Hematology
Sandhya R Panch, Thejaswi Bikkani, Vanessa Vargas, Jolynn Procter, James W Atkins, Virginia Guptill, Karen M Frank, Anna F Lau, David F Stroncek
BACKGROUND: Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products which fail release criteria (due to potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies...
August 8, 2018: Biology of Blood and Marrow Transplantation
Jaśmina Arabska, Anna Łucka, Dominik Strzelecki, Adam Wysokiński
AIM: Neurotrophin-3 (NT-3) is a neurotrophic factor responsible for promoting development, survival and function of neurons. NT-3 may be involved in the etiopathology of schizophrenia and mood disorders. However it must be cleared up if changes of NT-3 level are associated with schizophrenia itself or are secondary to certain symptoms (e.g. negative or depressive). The aim of this study was to examine whether the presence of negative or depressive symptoms affects peripheral NT-3 concentration in patients with schizophrenia...
August 8, 2018: Neuroscience Letters
N Golender, V Y Bumbarov, O Erster, M Beer, Y Khinich, K Wernike
Simbu serogroup viruses induce acute clinical diseases and abnormal courses of pregnancies in livestock. In Israel, two members of this serogroup, namely Akabane virus (AKAV) and Shuni virus (SHUV), were recently detected and, in Europe, Schmallenberg virus (SBV) poses a threat to ruminants. To address this emerging problem, a universal S-segment-based real-time RT-PCR assay (Uni-S) for the detection of Simbu serogroup viruses was established, which, additionally, enabled species identification of the detected viruses by subsequent sequencing...
August 8, 2018: Journal of Virological Methods
Gianni Dal Negro, Chantra Eskes, Susanne Belz, Caroline Bertein, Magda Chlebus, Marco Corvaro, Raffaella Corvi, Stephane Dhalluin, Marlies Halder, Jim Harvey, Martina Herman, Simone Hoffmann-Dörr, Karin Kilian, Denis Lambrigts, Charles Laroche, Susanna Louhimies, Catherine Mahony, Irene Manou, Pauline McNamee, Pilar Prieto, Kirsty Reid, Erwin Roggen, Katrin Schutte, Catrina Stirling, Sylvie Uhlrich, Renate Weissenhorn, Maurice Whelan
EU regulations call for the use of alternative methods to animal testing. During the last decade, an increasing number of alternative approaches have been formally adopted. In parallel, new 3Rs-relevant technologies and mechanistic approaches have increasingly contributed to hazard identification and risk assessment evolution. In this changing landscape, an EPAA meeting reviewed the challenges that different industry sectors face in the implementation of alternative methods following a science-driven approach...
August 8, 2018: Regulatory Toxicology and Pharmacology: RTP
Omid Tavassoly, Dorin Sade, Santu Bera, Shira Shaham-Niv, David Vocadlo, Ehud Gazit
Quinolinic acid (QA), a downstream neurometabolite in the kynurenine pathway, the biosynthetic pathway of tryptophan, is associated with neurodegenerative diseases pathology. Mutations in genes encoding kynurenine pathway enzymes, that control the level of QA production, are linked with elevated risk of developing Parkinson's disease (PD). Recent findings have revealed the accumulation and deposition of QA in post-mortem samples, as well as in cellular models of Alzheimer's disease (AD) and related disorders...
August 8, 2018: Journal of Molecular Biology
David C LaFon, Moon H Nahm
Quantitative assays that measure immune response to pneumococcal vaccines are not only important for the evaluation of vaccine immunogenicity and efficacy, but are also utilized in the clinical diagnosis of immune deficiency syndromes. Analytical methods have progressed in order to meet changing demands in both of these areas, from early methods to ELISA, and most recently multiplex bead array assays and opsonophagocytosis assays (OPA). It is necessary to understand the evolution of such techniques and the criteria for their interpretation in order to better inform the application of currently available methods, and to guide future investigation into assay development...
August 8, 2018: Journal of Immunological Methods
Rubén Martín-Escolano, Benjamín Aguilera-Venegas, Clotilde Marín, Álvaro Martín-Montes, Javier Martín-Escolano, Encarnación Medina-Carmona, Vicente J Arán, Manuel Sánchez-Moreno
Neglected tropical Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening disease that affects 5-8 million people in Latin America, and more than 10 million people worldwide. It is characterised by an acute phase, which is partly resolved by the immune system, but then develops as a chronic disease without an effective treatment. There is an urgent need for new antiprotozoal agents since the current standard therapeutic options, based on benznidazole and nifurtimox, are characterized by limited efficacy, toxicity and frequent failures in the treatment...
August 11, 2018: ChemMedChem
Sayan Ghosh, Peng Shang, Hiroto Terasaki, Nadezda Stepicheva, Stacey Hose, Meysam Yazdankhah, Joseph Weiss, Taiji Sakamoto, Imran A Bhutto, Shuli Xia, J Samuel Zigler, Ram Kannan, Jiang Qian, James T Handa, Debasish Sinha
Purpose: The RPE cells have a major role in the development of dry age-related macular degeneration (AMD). We present novel evidence that βA3/A1-crystallin, encoded by the Cryba1 gene, a protein known to be important for lysosomal clearance in the RPE, also has a role in epithelial-to-mesenchymal transition (EMT) of RPE cells. Methods: RPE from dry AMD globes, genetically engineered mice lacking Cryba1 globally or specifically in the RPE, spontaneous mutant rats (Nuc1) with a loss-of-function mutation in Cryba1, and the melanoma OCM3 cell line were used...
March 20, 2018: Investigative Ophthalmology & Visual Science
Agustina Bernal-Vicente, Daniel Cantabella, José Antonio Hernández, Pedro Diaz-Vivancos
In a previous work, we observed that mandelonitrile (MD), which controls cyanogenic glycoside turnover, is involved in salicylic acid (SA) biosynthesis in peach plants. In order to gain knowledge about the possible roles of this SA biosynthetic pathway, this current study looks at the effect of MD and phenylalanine (MD precursor) treatments on peach plant performance from an agronomic point of view. Abiotic (2g/L NaCl) and biotic (Plum pox virus) stresses were assayed. We recorded the following chlorophyll fluorescence parameters: quantum yield of photochemical energy conversion in PSII [Y(II)], photochemical quenching (qP) and the quantum yield of regulated non-photochemical energy loss in PSII and its coefficient [Y(NPQ) and qN]...
August 10, 2018: Plant Biology
Teemu Haikarainen, Sudarshan Murthy, Mirko M Maksimainen, Lari Lehtiö
Mono-ADP-ribosyltransferases of the PARP/ARTD enzyme family are enzymes catalyzing the transfer of a single ADP-ribose unit to target proteins. The enzymes have various roles in vital cellular processes such as DNA repair and transcription, and many of the enzymes are linked to cancer-relevant functions. Thus inhibition of the enzymes is a potential way to discover and develop new drugs against cancer. Here we describe an activity-based screening assay for mono-ADP-ribosyltransferases. The assay utilizes the natural substrate of the enzymes, NAD+ , and it is based on chemically converting the leftover substrate to a fluorophore and measuring its relative concentration after the enzymatic reaction...
2018: Methods in Molecular Biology
Jun Young Hong, Xiaoyu Zhang, Hening Lin
Sirtuins are a class of enzymes that utilize nicotinamide adenine dinucleotide, NAD+ , to remove various acyl groups from protein lysine residues. They have important biological functions and regulate numerous biological pathways. Small molecules that can modulate sirtuin enzymatic activities are potential therapeutic candidates to treat various human diseases. This protocol describes a high-performance liquid chromatography (HPLC)-based method to measure the enzyme kinetics for SIRT2 and SIRT6's demyristoylase activities and SIRT5's desuccinylase activity...
2018: Methods in Molecular Biology
Jeannette Abplanalp, Ann-Katrin Hopp, Michael O Hottiger
Despite substantial progress in ADP-ribosylation research in recent years, the identification of ADP-ribosylated proteins, their ADP-ribose acceptors sites, and the respective writers and erasers remains challenging. The use of recently developed mass spectrometric methods helps to further characterize the ADP-ribosylome and its regulatory enzymes under different conditions and in different cell types. Validation of these findings may be achieved by in vitro assays for the respective enzymes. In the below method, we describe how recombinant ADP-ribosylated proteins are demodified in vitro with mono-ADP-ribosylhydrolases of choice to elucidate substrate and potentially also site specificity of these enzymes...
2018: Methods in Molecular Biology
Giovanna Grimaldi, Giuliana Catara, Carmen Valente, Daniela Corda
ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification...
2018: Methods in Molecular Biology
Yi X Chan, Matthew W Knuiman, Mark L Divitini, David J Handelsman, John P Beilby, Bu B Yeap
Androgens, notably testosterone (T), have been implicated in development of several common cancers and prostate cancer; however, precise mechanisms remain unclear. This study assessed prospective associations of serum T, dihydrotestosterone (DHT) and estradiol (E2) with overall cancer (excluding skin cancer), prostate, colorectal and lung cancer risk in 1574 community-dwelling men aged 25-84 years. Sex hormones were assayed using mass spectrometry and men were followed for 20 years with outcomes ascertained using data linkage...
August 10, 2018: Hormones & Cancer
Alok K Shah, Gunter Hartel, Ian Brown, Clay Winterford, Renhua Na, Kim-Anh Lê Cao, Bradley A Spicer, Michelle A Dunstone, Wayne A Phillips, Reginald V Lord, Andrew P Barbour, David I Watson, Virendra Joshi, David C Whiteman, Michelle M Hill
Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett's esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance for BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. Serum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and USA (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier 3 assay...
August 10, 2018: Molecular & Cellular Proteomics: MCP
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