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High-Throughput, Volume 6, Issue 4 (December 2017)

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Research

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Open AccessArticle Development of a Bead-Based Multiplex Assay for the Analysis of the Serological Response against the Six Pathogens HAV, HBV, HCV, CMV, T. gondii, and H. pylori
High-Throughput 2017, 6(4), 14; doi:10.3390/ht6040014
Received: 31 August 2017 / Revised: 17 October 2017 / Accepted: 26 October 2017 / Published: 30 October 2017
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Abstract
The spread of infectious diseases and vaccination history are common subjects of epidemiological and immunological research studies. Multiplexed serological assays are useful tools for assessing both current and previous infections as well as vaccination efficacy. We developed a serological multi-pathogen assay for hepatitis
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The spread of infectious diseases and vaccination history are common subjects of epidemiological and immunological research studies. Multiplexed serological assays are useful tools for assessing both current and previous infections as well as vaccination efficacy. We developed a serological multi-pathogen assay for hepatitis A, B and C virus, cytomegalovirus (CMV), Toxoplasma gondii, and Helicobacter pylori using a bead-based multiplex assay format. The multi-pathogen assay consisting of 15 antigens was utilized for the analysis of the serological response in elderly individuals of an influenza vaccination study (n = 34). The technical assay validation revealed a mean intra-assay precision of coefficient of variation (CV) = 3.2 ± 1.5% and a mean inter-assay precision of CV = 8.2 ± 5.3% across all 15 antigens and all tested samples, indicating a robust test system. Furthermore, the assay shows high sensitivities (ranging between 94% and 100%) and specificities (ranging between 93% and 100%) for the different pathogens. The highest seroprevalence rates in our cohort were observed for hepatitis A virus (HAV; 73.5%), followed by CMV (70.6%), T. gondii (67.6%) and H. pylori (32.4%). Seroprevalences for hepatitis B virus (HBV, 8.8%) and hepatitis C virus (HCV, 0%) were low. The seroprevalences observed in our study were similar to those from other population-based studies in Germany. In summary, we conclude that our multiplex serological assay represents a suitable tool for epidemiological studies. Full article
(This article belongs to the Special Issue Protein Microarrays)
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Open AccessArticle Study of the Humoral Immune Response towards HCV Genotype 4 Using a Bead-Based Multiplex Serological Assay
High-Throughput 2017, 6(4), 15; doi:10.3390/ht6040015
Received: 31 August 2017 / Revised: 26 September 2017 / Accepted: 23 October 2017 / Published: 30 October 2017
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Abstract
Hepatitis C is one of the leading causes of hepatocellular carcinoma and remains at a high prevalence in Egypt and other resource-limited countries. Several hepatitis C virus (HCV) genotypes are distributed throughout the world, with genotype 4 being most common in North and
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Hepatitis C is one of the leading causes of hepatocellular carcinoma and remains at a high prevalence in Egypt and other resource-limited countries. Several hepatitis C virus (HCV) genotypes are distributed throughout the world, with genotype 4 being most common in North and Central Africa. We developed a multiplex serological assay for the detection of the HCV specific humoral immune response, with a focus on genotype 4. For the multiplex HCV assay we used twelve antigenic regions of different HCV proteins (core, and non-structural (NS) proteins NS3, NS4, NS5A, NS5B) and validated the assay technically and clinically. In comparison to a commercially available test, our assay revealed a higher sensitivity for genotype 4, and is therefore more suited for studying immune seroconversion in samples from acutely infected Egyptian HCV patients. Furthermore, our assay discriminates acutely and chronically infected HCV patients. Of 296 well characterized HCV patient samples, 83.9% of the acute samples and 86.5% of the chronic samples could be correctly classified. In sum, this newly developed serological HCV assay has a higher sensitivity for HCV genotype 4, and can thus improve diagnostic accuracy. Through the discrimination of acutely and chronically infected HCV patients the assay may be useful in supporting clinical management of HCV patients. Full article
(This article belongs to the Special Issue Protein Microarrays)
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Open AccessArticle A Computational Workflow Translates a 58-Gene Signature to a Formalin-Fixed, Paraffin-Embedded Sample-Based Companion Diagnostic for Personalized Treatment of the BRAF-Mutation-Like Subtype of Colorectal Cancers
High-Throughput 2017, 6(4), 16; doi:10.3390/ht6040016
Received: 12 October 2017 / Revised: 27 October 2017 / Accepted: 30 October 2017 / Published: 6 November 2017
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Abstract
Colorectal cancer patients with the BRAF(p.V600E) mutation have poor prognosis in metastatic setting. Personalized treatment options and companion diagnostics are needed to better treat these patients. Previously, we developed a 58-gene signature to characterize the distinct gene expression pattern of BRAF-mutation-like
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Colorectal cancer patients with the BRAF(p.V600E) mutation have poor prognosis in metastatic setting. Personalized treatment options and companion diagnostics are needed to better treat these patients. Previously, we developed a 58-gene signature to characterize the distinct gene expression pattern of BRAF-mutation-like subtype (accuracy 91.1%). Further experiments repurposed drug Vinorelbine as specifically lethal to this BRAF-mutation-like subtype. The aim of this study is to translate this 58-gene signature from a research setting to a robust companion diagnostic that can use formalin-fixed, paraffin-embedded (FFPE) samples to select patients with the BRAF-mutation-like subtype. BRAF mutation and gene expression data of 302 FFPE samples were measured (mutants = 57, wild-type = 245). The performance of the 58-gene signature in FFPE samples showed a high sensitivity of 89.5%. In the identified BRAF-mutation-like subtype group, 50% of tumours were known BRAF mutants, and 50% were BRAF wild-type. The stability of the 58-gene signature in FFPE samples was evaluated by two control samples over 40 independent experiments. The standard deviations (SD) were within the predefined criteria (control 1: SD = 0.091, SD/Range = 3.0%; control 2: SD = 0.169, SD/Range = 5.5%). The fresh frozen version and translated FFPE version of this 58-gene signature were compared using 170 paired fresh frozen and FFPE samples and the result showed high consistency (agreement = 99.3%). In conclusion, we translated this 58-gene signature to a robust companion diagnostic that can use FFPE samples. Full article
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Open AccessArticle Application of High-Throughput Assays to Examine Phospho-Modulation of the Late Steps of Regulated Exocytosis
High-Throughput 2017, 6(4), 17; doi:10.3390/ht6040017
Received: 20 September 2017 / Revised: 1 November 2017 / Accepted: 6 November 2017 / Published: 13 November 2017
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Abstract
Abstract: Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca2+-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical
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Abstract: Regulated exocytosis enables a range of physiological functions including neurotransmission, and the late steps (i.e., docking, priming and Ca2+-triggered membrane fusion) are modulated by a highly conserved set of proteins and lipids. Many of the molecular components and biochemical interactions required have been identified; the precise mechanistic steps they modulate and the biochemical interactions that need to occur across steps are still the subject of intense investigation. Particularly, although the involvement of phosphorylation in modulating exocytosis has been intensively investigated over the past three decades, it is unclear which phosphorylation events are a conserved part of the fundamental fusion mechanism and/or serve as part of the physiological fusion machine (e.g., to modulate Ca2+ sensitivity). Here, the homotypic fusion of cortical vesicles was monitored by utilizing new high-throughput, cost-effective assays to assess the influence of 17 small molecule phospho-modulators on docking/priming, Ca2+ sensitivity and membrane fusion. Specific phosphatases and casein kinase 2 are implicated in modulating the Ca2+ sensitivity of fusion, whereas sphingosine kinase is implicated in modulating the ability of vesicles to fuse. These results indicate the presence of multiple kinases and phosphatases on the vesicles and critical phosphorylation sites on vesicle membrane proteins and lipids that directly influence late steps of regulated exocytosis. Full article
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Open AccessArticle Development and Validation of an Ultrasensitive Procalcitonin Sandwich Immunoassay
High-Throughput 2017, 6(4), 18; doi:10.3390/ht6040018
Received: 20 October 2017 / Revised: 8 November 2017 / Accepted: 13 November 2017 / Published: 16 November 2017
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Abstract
Procalcitonin (PCT) is well established as a highly specific biomarker for the detection of bacterial infections and sepsis. However, the currently available diagnostic tests are not able to detect very low or very early increases of PCT or even baseline levels in healthy
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Procalcitonin (PCT) is well established as a highly specific biomarker for the detection of bacterial infections and sepsis. However, the currently available diagnostic tests are not able to detect very low or very early increases of PCT or even baseline levels in healthy individuals or patients with non-bacterial infections. In order to be able to detect these very low concentrations of PCT, a sandwich immunoassay was developed using high sensitivity Single Molecule Array technology (Simoa). The assay was thoroughly validated and applied to analyze human cerebrospinal fluid (CSF) and serum samples from patients with bacterial or viral meningitis as well as CSF, serum, and K2 EDTA plasma from healthy control subjects. A 50-fold increase in sensitivity compared to the current gold standard assays was achieved, which was sensitive enough for the detection of baseline PCT levels. Both serum and CSF showed significantly elevated PCT levels in patients with bacterial meningitis compared to patients with viral meningitis and the healthy control group. Procalcitonin concentration levels for patients with viral meningitis and the control group could be measured, but were not significantly different. The determination of PCT in the low pg·mL−1 range could help to improve the monitoring of bacterial infectious diseases, as PCT level changes could be detected earlier. Full article
(This article belongs to the Special Issue Protein Microarrays)
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Review

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Open AccessReview Small RNAs in Circulating Exosomes of Cancer Patients: A Minireview
High-Throughput 2017, 6(4), 13; doi:10.3390/ht6040013
Received: 30 August 2017 / Revised: 18 September 2017 / Accepted: 29 September 2017 / Published: 6 October 2017
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Abstract
Extracellular vesicles (EVs) secreted from many cell types play important roles in intercellular communication, both as paracrine and endocrine factors, as they can circulate in biological fluids, including plasma. Amid EVs, exosomes are actively secreted vesicles that contain proteins, lipids, soluble factors, and
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Extracellular vesicles (EVs) secreted from many cell types play important roles in intercellular communication, both as paracrine and endocrine factors, as they can circulate in biological fluids, including plasma. Amid EVs, exosomes are actively secreted vesicles that contain proteins, lipids, soluble factors, and nucleic acids, including microRNAs (miRNAs) and other classes of small RNAs (sRNA). miRNAs are prominent post‐transcriptional regulators of gene expression and epigenetic silencers of transcription. We concisely review the roles of miRNAs in cell‐fate determination and development and their regulatory activity on almost all the processes and pathways controlling tumor formation and progression. Next, we consider the evidence linking exosomes to tumor progression, particularly to the setting‐up of permissive pre‐metastatic niches. The study of exosomes in patients with different survival and therapy response can inform on the possible correlations between exosomal cargo and disease features. Moreover, the exploration of circulating exosomes as possible sources of non‐invasive biomarkers could give new implements for anti‐cancer therapy and metastasis prevention. Since the characterization of sRNAs in exosomes of cancer patients sparks opportunities to better understand their roles in cancer, we briefly present current experimental and computational protocols for sRNAs analysis in circulating exosomes by RNA‐seq. Full article
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