High-Throughput — Open Access Journal

Pre-transfusion bedside compatibility test is mandatory to check that the donor and the recipient present compatible groups before any transfusion is performed. Although blood typing devices are present on the market, they still suffer from various drawbacks, like results that are based on naked-eye observation or difficulties in blood handling and process automation. In this study, we addressed the development of a red blood cells (RBC) agglutination assay for point-of-care blood typing. An injection molded microfluidic chip that is designed to enhance capillary flow contained anti-A or anti-B dried reagents inside its microchannel. The only blood handling step in the assay protocol consisted in the deposit of a blood drop at the tip of the biochip, and imaging was then achieved. The embedded reagents were able to trigger RBC agglutination in situ, allowing for us to monitor in real time the whole process. An image processing algorithm was developed on diluted bloods to compute real-time agglutination indicator and was further validated on undiluted blood. Through this proof of concept, we achieved efficient, automated, real time, and quantitative measurement of agglutination inside a passive biochip for blood typing which could be further generalized to blood biomarker detection and quantification. Full article

Metabolomics has made significant progress in multiple fronts in the last 18 months. This minireview aimed to give an overview of these advancements in the light of their contribution to targeted and untargeted metabolomics. New computational approaches have emerged to overcome the manual absolute quantitation step of metabolites in one-dimensional (1D) ¹H nuclear magnetic resonance (NMR) spectra. This provides more consistency between inter-laboratory comparisons. Integration of two-dimensional (2D) NMR metabolomics databases under a unified web server allowed for very accurate identification of the metabolites that have been catalogued in these databases. For the remaining uncatalogued and unknown metabolites, new cheminformatics approaches have been developed by combining NMR and mass spectrometry (MS). These hybrid MS/NMR approaches accelerated the identification of unknowns in untargeted studies, and now they are allowing for profiling ever larger number of metabolites in application studies. Full article

Recent and rapid technological advances in molecular sciences have dramatically increased the ability to carry out high-throughput studies characterized by big data production. This, in turn, led to the consequent negative effect of highlighting the presence of a gap between data yield and their analysis. Indeed, big data management is becoming an increasingly important aspect of many fields of molecular research including the study of human diseases. Now, the challenge is to identify, within the huge amount of data obtained, that which is of clinical relevance. In this context, issues related to data interpretation, sharing and storage need to be assessed and standardized. Once this is achieved, the integration of data from different -omic approaches will improve the diagnosis, monitoring and therapy of diseases by allowing the identification of novel, potentially actionably biomarkers in view of personalized medicine. Full article

Over the last three decades, novel “omics” platform technologies for the sequencing of DNA and complementary DNA (cDNA) (RNA-Seq), as well as for the analysis of proteins and metabolites by mass spectrometry, have become more and more available and increasingly found their way into general laboratory life. With this, the ability to generate highly multivariate datasets on the biological systems of choice has increased tremendously. However, the processing and, perhaps even more importantly, the integration of “omics” datasets still remains a bottleneck, although considerable computational and algorithmic advances have been made in recent years. In this mini-review, we use a number of recent “multi-omics” approaches realized in our laboratories as a common theme to discuss possible pitfalls of applying “omics” approaches and to highlight some useful tools for data integration and visualization in the form of an exemplified case study. In the selected example, we used a combination of transcriptomics and metabolomics alongside phenotypic analyses to functionally characterize a small number of Cycling Dof Transcription Factors (CDFs). It has to be remarked that, even though this approach is broadly used, the given workflow is only one of plenty possible ways to characterize target proteins. Full article

The progress of cancer genome sequencing projects yields unprecedented information of mutations for numerous patients. However, the complexity of mutation profiles of cancer patients hinders the further understanding to mechanisms of oncogenesis. One basic question is how to find mutations with functional impacts. In this work, we introduce a computational method to predict functional somatic mutations of each patient by integrating mutation recurrence with expression profile similarity. With this method, the functional mutations are determined by checking the mutation enrichment among a group of patients with similar expression profiles. We applied this method to three cancer types and identified the functional mutations. Comparison of the predictions for three cancer types suggested that most of the functional mutations were cancer-type-specific with one exception to p53. By checking predicted results, we found that our method effectively filtered non-functional mutations resulting from large protein sizes. In addition, this method can also perform functional annotation to each patient to describe their association with signalling pathways or biological processes. In breast cancer, we predicted “cell adhesion” and other terms to be significantly associated with oncogenesis. Full article

Among applicable high-throughput techniques in cardiovascular biology, whole-transcriptome sequencing is of particular use. By utilizing RNA that is isolated from virtually all cells and tissues, the entire transcriptome can be evaluated. In comparison with other high-throughput approaches, RNA sequencing is characterized by a relatively low-cost and large data output, which permits a comprehensive analysis of spatiotemporal variation in the gene expression profile. Both shear stress and cyclic strain exert hemodynamic force upon the arterial endothelium and are considered to be crucial determinants of endothelial physiology. Laminar blood flow results in a high shear stress that promotes atheroresistant endothelial phenotype, while a turbulent, oscillatory flow yields a pathologically low shear stress that disturbs endothelial homeostasis, making respective arterial segments prone to atherosclerosis. Severe atherosclerosis significantly impairs blood supply to the organs and frequently requires bypass surgery or an arterial replacement surgery that requires tissue-engineered vascular grafts. To provide insight into patterns of gene expression in endothelial cells in native or bioartificial arteries under different biomechanical conditions, this article discusses applications of whole-transcriptome sequencing in endothelial mechanobiology and vascular tissue engineering. Full article

Drug discovery encompasses processes ranging from target selection and validation to the selection of a development candidate. While comprehensive drug discovery work flows are implemented predominantly in the big pharma domain, early discovery focus in academia serves to identify probe molecules that can serve as tools to study targets or pathways. Despite differences in the ultimate goals of the private and academic sectors, the same basic principles define the best practices in early discovery research. A successful early discovery program is built on strong target definition and validation using a diverse set of biochemical and cell-based assays with functional relevance to the biological system being studied. The chemicals identified as hits undergo extensive scaffold optimization and are characterized for their target specificity and off-target effects in in vitro and in animal models. While the active compounds from screening campaigns pass through highly stringent chemical and Absorption, Distribution, Metabolism, and Excretion (ADME) filters for lead identification, the probe discovery involves limited medicinal chemistry optimization. The goal of probe discovery is identification of a compound with sub-µM activity and reasonable selectivity in the context of the target being studied. The compounds identified from probe discovery can also serve as starting scaffolds for lead optimization studies. Full article

Very often the clinical features of rare neurodegenerative disorders overlap with those of other, more common clinical disturbances. As a consequence, not only the true incidence of these disorders is underestimated, but many patients also experience a significant delay before a definitive diagnosis. Under this scenario, it appears clear that any accurate tool producing information about the pathological mechanisms of these disorders would offer a novel context for their precise identification by strongly enhancing the interpretation of symptoms. With the advent of proteomics, detection and identification of proteins in different organs/tissues, aimed at understanding whether they represent an attractive tool for monitoring alterations in these districts, has become an area of increasing interest. The aim of this report is to provide an overview of the most recent applications of proteomics as a new strategy for identifying biomarkers with a clinical utility for the investigation of rare neurodegenerative disorders. Full article

Although a number of combinatorial/high-throughput approaches have been developed for biomaterial hydrogel optimization, a gradient sample approach is particularly well suited to identify hydrogel property thresholds that alter cellular behavior in response to interacting with the hydrogel due to reduced variation in material preparation and the ability to screen biological response over a range instead of discrete samples each containing only one condition. This review highlights recent work on cell–hydrogel interactions using a gradient material sample approach. Fabrication strategies for composition, material and mechanical property, and bioactive signaling gradient hydrogels that can be used to examine cell–hydrogel interactions will be discussed. The effects of gradients in hydrogel samples on cellular adhesion, migration, proliferation, and differentiation will then be examined, providing an assessment of the current state of the field and the potential of wider use of the gradient sample approach to accelerate our understanding of matrices on cellular behavior. Full article

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

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

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

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

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

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

Increasing evidence suggests that the numbers of long non‐coding RNAs (lncRNAs) are more than those of protein‐coding genes in various organisms. Although the detection methods for lncRNAs are being increasingly established, there are advantages and disadvantages that exist for each method. In this opinion article, I highlight the differences between microarrays and RNA sequencing (RNA‐seq) for the detection of lncRNAs. Compared to RNA‐seq, microarrays are limited to the known sequences. However, the detection method as well as data analysis workflow is more established, which makes it easier to analyze the data for bench scientists without extensive knowledge about computer programming. In order to highlight the usage of microarrays over RNA‐seq for the detection of lncRNAs, we are organizing a special issue for High‐Throughput called “Microarrays in Non‐Coding RNAs Profiling”, which will include the specific usages of microarrays for lncRNAs. Full article

This study aims to consider the microbial distribution in oral disease, as well as gene analysis and expression, in elucidating: 1, the fundamental underpinnings of oral disease, and 2, the potential relationship between oral diseases and systemic health. A key focus is identifying the microbiota associated with oral disease manifestations characterized by both conventional microbiological and molecular methods. Variations in the observed microbial populations characterized by conventional and molecular approaches have been identified for caries, periodontitis, peri-implantitis, and stomatitis. The discovery of therapeutic approaches for oral disease will require comprehensive microbial and genomic analysis. This study evaluated the current state of the relevant microbial and genomic information for several prevalent oral diseases. Full article