Correction: Gemoll et al. Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences after Differential Ultracentrifugation and ExoQuick™ Isolation. J. Clin. Med. 2020, 9, 1429.

The authors wish to make the following corrections to this paper [...].

hsa-miR-374b-5p regulates expression of the gene U2AF homology motif (UHM) kinase 1.

OBJECTIVE: We aimed to identify a microRNA (miRNA) that is significantly upregulated in blood and in cells of the oral mucosa upon exposure to the periodontitis main risk factors oral inflammation and tobacco smoke, to subsequently identify its target gene and to describe the molecular mechanism of gene regulation. BACKGROUND: miRNAs are associated with many disorders. Array-based miRNA expression studies indicated a number of differentially expressed miRNAs in the pathology of oral diseases. However, these miRNAs mostly lacked replication, and their target genes have remained unknown. METHODS: 863 miRNAs were analyzed in blood from 18 PD cases and 70 controls (Geniom Biochip). Selected miRNAs were analyzed for upregulation in the inflamed oral mucosa of PD patients using published miRNA expression profiling studies from gingival cells. hsa-miR-374b-5p mimic was overexpressed in primary gingival fibroblasts (pGFs) from 3 donors, and genome-wide mRNA expression was quantified (Clarion Array). Gene-specific regulation was validated by qRT-PCR and Luciferase activity in HeLa cells. RESULTS: hsa-miR-374b-5p showed >twofold change (FC) in 3 independent studies performed in blood, gingival tissues, and cells. After hsa-miR-374b-5p overexpression, genome-wide expression analysis showed UHMK1 as top 1 downregulated gene in pGFs (p = 2.5 × 10-04 , fold change = -1.8). Reporter genes demonstrated that hsa-miR-374b-5p downregulates mRNA levels (p = .02; FC = -1.5), leading to reduction in protein activity (p = .013, FC = -1.3). CONCLUSIONS: hsa-miR-374b-5p is upregulated in blood and ginvial cells exposed to oral inflammation and tobacco smoke and regulates UHMK1, which has a role in osteoclast differentiation.

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Publisher Correction: Identification and characterization of two functional variants in the human longevity gene FOXO3.

The original version of this Article contained an error in the spelling of the author Robert Häsler, which was incorrectly given as Robert Häesler. This has now been corrected in both the PDF and HTML versions of the Article.

Plasmonic Metaparticles on a Blackbody Create Vivid Reflective Colors for Naked-Eye Environmental and Clinical Biodetection.

Plasmonic dipoles are famous for their strong absorptivity rather than their reflectivity. Here, the as-yet unknown specular reflection and the Brewster effect of ultrafine plasmonic dipoles, metaparticles, are introduced and exploited as the basis of new design rules for advanced applications. A configuration of "Plasmonic metaparticles on a blackbody" is demonstrated and utilized for the design of a tailored perfect-colored absorber and for visual detection of environmental dielectrics that is not readily done by extinction plasmonics. Moreover, the Plasmonic Brewster Wavelength (PBW) effect is introduced as a new platform for the naked-eye and bulk biodetection of analytes. The technique operates based on slight changes of molecular polarizability which is not detectable via conventional plasmon resonance techniques. As a specific highlight, the clinical applicability of the PBW method is demonstrated while addressing the transduction plasmonic techniques' challenge in detection of bulk refractive index changes of the healthy and diseased human serum exosomes. Finally, the sputtering-based fabrication method used here is simple, inexpensive, and scalable, and does not require the sophisticated patterning approach of lithography or precise alignment of light coupling for the biodetection.

Identification and characterization of two functional variants in the human longevity gene FOXO3.

FOXO3 is consistently annotated as a human longevity gene. However, functional variants and underlying mechanisms for the association remain unknown. Here, we perform resequencing of the FOXO3 locus and single-nucleotide variant (SNV) genotyping in three European populations. We find two FOXO3 SNVs, rs12206094 and rs4946935, to be most significantly associated with longevity and further characterize them functionally. We experimentally validate the in silico predicted allele-dependent binding of transcription factors (CTCF, SRF) to the SNVs. Specifically, in luciferase reporter assays, the longevity alleles of both variants show considerable enhancer activities that are reversed by IGF-1 treatment. An eQTL database search reveals that the alleles are also associated with higher FOXO3 mRNA expression in various human tissues, which is in line with observations in long-lived model organisms. In summary, we present experimental evidence for a functional link between common intronic variants in FOXO3 and human longevity.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.

Concentration of circulating miRNA-containing particles in serum enhances miRNA detection and reflects CRC tissue-related deregulations.

The emerging potential of miRNAs as biomarkers for cancer detection demands parallel evaluation of strategies for reliable identification of disease-related signatures from easily accessible and pertinent body compartments. Here, we addressed whether efficient concentration of circulating miRNA-carrying particles is a rationale for miRNA biomarker discovery. We systematically compared miRNA signatures in 93 RNA preparations from three serum entities (whole serum, particle-concentrated, and particle-depleted fractions) and corresponding tissue samples from patients with colorectal cancer (CRC) as a model disease. Significant differences between whole sera and particle-concentrated serum fractions of CRC patients emerged for 45 of 742 tested miRNAs. Twenty-eight of these 45 miRNAs were differentially expressed between particle-concentrated serum fractions of metastatic CRC- and healthy individuals. Over half of these candidates (15 of 28) showed deregulations only in concentrated serum fractions, but not in whole sera, compared to the respective controls.Our results also provided evidence of a consistent downregulation of miR-486 and miR-92a, and further showed a possible "strand-specific" deregulation of extracellular miRNAs in CRC. More importantly, most of the identified miRNAs in the enriched sera reflected the patterns of the corresponding tumor tissues and showed links to cancer-related inflammation. Further investigation of seven serum pools revealed a subset of potential extracellular miRNA candidates to be implicated in both neoplastic and inflammatory bowel disease.Our findings demonstrate that enrichment and sensitive detection of miRNA carriers is a promising approach to detect CRC-related pathological changes in liquid biopsies, and has potential for clinical diagnostics.

Targeted Resequencing and Functional Testing Identifies Low-Frequency Missense Variants in the Gene Encoding GARP as Significant Contributors to Atopic Dermatitis Risk.

Gene-mapping studies have consistently identified a susceptibility locus for atopic dermatitis and other inflammatory diseases on chromosome band 11q13.5, with the strongest association observed for a common variant located in an intergenic region between the two annotated genes C11orf30 and LRRC32. Using a targeted resequencing approach we identified low-frequency and rare missense mutations within the LRRC32 gene encoding the protein GARP, a receptor on activated regulatory T cells that binds latent transforming growth factor-ß. Subsequent association testing in more than 2,000 atopic dermatitis patients and 2,000 control subjects showed a significant excess of these LRRC32 variants in individuals with atopic dermatitis. Structural protein modeling and bioinformatic analysis predicted a disruption of protein transport upon these variants, and overexpression assays in CD4+CD25- T cells showed a significant reduction in surface expression of the mutated protein. Consistently, flow cytometric (FACS) analyses of different T-cell subtypes obtained from atopic dermatitis patients showed a significantly reduced surface expression of GARP and a reduced conversion of CD4+CD25- T cells into regulatory T cells, along with lower expression of latency-associated protein upon stimulation in carriers of the LRRC32 A407T variant. These results link inherited disturbances of transforming growth factor-ß signaling with atopic dermatitis risk.

New technologies for DNA analysis--a review of the READNA Project.

The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.

Sparse Modeling Reveals miRNA Signatures for Diagnostics of Inflammatory Bowel Disease.

The diagnosis of inflammatory bowel disease (IBD) still remains a clinical challenge and the most accurate diagnostic procedure is a combination of clinical tests including invasive endoscopy. In this study we evaluated whether systematic miRNA expression profiling, in conjunction with machine learning techniques, is suitable as a non-invasive test for the major IBD phenotypes (Crohn's disease (CD) and ulcerative colitis (UC)). Based on microarray technology, expression levels of 863 miRNAs were determined for whole blood samples from 40 CD and 36 UC patients and compared to data from 38 healthy controls (HC). To further discriminate between disease-specific and general inflammation we included miRNA expression data from other inflammatory diseases (inflammation controls (IC): 24 chronic obstructive pulmonary disease (COPD), 23 multiple sclerosis, 38 pancreatitis and 45 sarcoidosis cases) as well as 70 healthy controls from previous studies. Classification problems considering 2, 3 or 4 groups were solved using different types of penalized support vector machines (SVMs). The resulting models were assessed regarding sparsity and performance and a subset was selected for further investigation. Measured by the area under the ROC curve (AUC) the corresponding median holdout-validated accuracy was estimated as ranging from 0.75 to 1.00 (including IC) and 0.89 to 0.98 (excluding IC), respectively. In combination, the corresponding models provide tools for the distinction of CD and UC as well as CD, UC and HC with expected classification error rates of 3.1 and 3.3%, respectively. These results were obtained by incorporating not more than 16 distinct miRNAs. Validated target genes of these miRNAs have been previously described as being related to IBD. For others we observed significant enrichment for IBD susceptibility loci identified in earlier GWAS. These results suggest that the proposed miRNA signature is of relevance for the etiology of IBD. Its diagnostic value, however, should be further evaluated in large, independent, clinically well characterized cohorts.

miRNAs can be generally associated with human pathologies as exemplified for miR-144.

BACKGROUND: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. METHODS: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. RESULTS: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 (95% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. CONCLUSIONS: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers.

From next-generation sequencing alignments to accurate comparison and validation of single-nucleotide variants: the pibase software.

Scientists working with single-nucleotide variants (SNVs), inferred by next-generation sequencing software, often need further information regarding true variants, artifacts and sequence coverage gaps. In clinical diagnostics, e.g. SNVs must usually be validated by visual inspection or several independent SNV-callers. We here demonstrate that 0.5-60% of relevant SNVs might not be detected due to coverage gaps, or might be misidentified. Even low error rates can overwhelm the true biological signal, especially in clinical diagnostics, in research comparing healthy with affected cells, in archaeogenetic dating or in forensics. For these reasons, we have developed a package called pibase, which is applicable to diploid and haploid genome, exome or targeted enrichment data. pibase extracts details on nucleotides from alignment files at user-specified coordinates and identifies reproducible genotypes, if present. In test cases pibase identifies genotypes at 99.98% specificity, 10-fold better than other tools. pibase also provides pair-wise comparisons between healthy and affected cells using nucleotide signals (10-fold more accurately than a genotype-based approach, as we show in our case study of monozygotic twins). This comparison tool also solves the problem of detecting allelic imbalance within heterozygous SNVs in copy number variation loci, or in heterogeneous tumor sequences.

Accurate variant detection across non-amplified and whole genome amplified DNA using targeted next generation sequencing.

BACKGROUND: Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. RESULTS: The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. CONCLUSION: Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing.

BACKGROUND: Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). RESULTS: We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent 'read-backmapping' to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach. CONCLUSIONS: We recommend applying our general 'two-step' mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results.

Genome-wide miRNA signatures of human longevity.

Little is known about the functions of miRNAs in human longevity. Here, we present the first genome-wide miRNA study in long-lived individuals (LLI) who are considered a model for healthy aging. Using a microarray with 863 miRNAs, we compared the expression profiles obtained from blood samples of 15 centenarians and nonagenarians (mean age 96.4 years) with those of 55 younger individuals (mean age 45.9 years). Eighty miRNAs showed aging-associated expression changes, with 16 miRNAs being up-regulated and 64 down-regulated in the LLI relative to the younger probands. Seven of the eight selected aging-related biomarkers were technically validated using quantitative RT-PCR, confirming the microarray data. Three of the eight miRNAs were further investigated in independent samples of 15 LLI and 17 younger participants (mean age 101.5 and 36.9 years, respectively). Our screening confirmed previously published miRNAs of human aging, thus reflecting the utility of the applied approach. The hierarchical clustering analysis of the miRNA microarray expression data revealed a distinct separation between the LLI and the younger controls (P-value < 10(-5) ). The down-regulated miRNAs appeared as a cluster and were more often reported in the context of diseases than the up-regulated miRNAs. Moreover, many of the differentially regulated miRNAs are known to exhibit contrasting expression patterns in major age-related diseases. Further in silico analyses showed enrichment of potential targets of the down-regulated miRNAs in p53 and other cancer pathways. Altogether, synchronized miRNA-p53 activities could be involved in the prevention of tumorigenesis and the maintenance of genomic integrity during aging.

Targeted enrichment of genomic DNA regions for next-generation sequencing.

In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.

Toward the blood-borne miRNome of human diseases.

In a multicenter study, we determined the expression profiles of 863 microRNAs by array analysis of 454 blood samples from human individuals with different cancers or noncancer diseases, and validated this 'miRNome' by quantitative real-time PCR. We detected consistently deregulated profiles for all tested diseases; pathway analysis confirmed disease association of the respective microRNAs. We observed significant correlations (P = 0.004) between the genomic location of disease-associated genetic variants and deregulated microRNAs.

Mutational characterization of the bile acid receptor TGR5 in primary sclerosing cholangitis.

BACKGROUND: TGR5, the G protein-coupled bile acid receptor 1 (GPBAR1), has been linked to inflammatory pathways as well as bile homeostasis, and could therefore be involved in primary sclerosing cholangitis (PSC) a chronic inflammatory bile duct disease. We aimed to extensively investigate TGR5 sequence variation in PSC, as well as functionally characterize detected variants. METHODOLOGY/PRINCIPAL FINDINGS: Complete resequencing of TGR5 was performed in 267 PSC patients and 274 healthy controls. Six nonsynonymous mutations were identified in addition to 16 other novel single-nucleotide polymorphisms. To investigate the impact from the nonsynonymous variants on TGR5, we created a receptor model, and introduced mutated TGR5 constructs into human epithelial cell lines. By using confocal microscopy, flow cytometry and a cAMP-sensitive luciferase assay, five of the nonsynonymous mutations (W83R, V178M, A217P, S272G and Q296X) were found to reduce or abolish TGR5 function. Fine-mapping of the previously reported PSC and UC associated locus at chromosome 2q35 in large patient panels revealed an overall association between the TGR5 single-nucleotide polymorphism rs11554825 and PSC (odds ratio = 1.14, 95% confidence interval: 1.03-1.26, p = 0.010) and UC (odds ratio = 1.19, 95% confidence interval 1.11-1.27, p = 8.5 x 10(-7)), but strong linkage disequilibrium precluded demarcation of TGR5 from neighboring genes. CONCLUSIONS/SIGNIFICANCE: Resequencing of TGR5 along with functional investigations of novel variants provided unique insight into an important candidate gene for several inflammatory and metabolic conditions. While significant TGR5 associations were detected in both UC and PSC, further studies are needed to conclusively define the role of TGR5 variation in these diseases.

Systematic evaluation of the effect of common SNPs on pre-mRNA splicing.

The evolutionary and biomedical importance of differential mRNA splicing is well established. Numerous studies have assessed patterns of differential splicing in different genes and correlated these patterns to the genotypes for adjacent single-nucleotide polymorphisms (SNPs). Here, we have chosen a reverse approach and screened dbSNP for common SNPs at either canonical splice sites or exonic splice enhancers (ESEs) that would be classified as putatively splicing-relevant by bioinformatic tools. The 223 candidate SNPs retrieved from dbSNP were experimentally tested using a previously established panel of 92 matching DNAs and cDNAs. For each SNP, 16 cDNAs providing a balanced representation of the genotypes at the respective SNP were investigated by nested RT-PCR and subsequent sequencing. Putative allele-dependent splicing was verified by the cloning of PCR products. The positive predictive value of the bioinformatics tools turned out to be low, ranging from 0% for ESEfinder to 9% (in the case of acceptor-site SNPs) for a recently reported neural network. The results highlight the need for a better understanding of the sequence characteristics of functional splice-sites to improve our ability to predict in silico the splicing relevance of empirically observed DNA sequence variants.