Healthy Control Serum Samples Research Articles

A Study on the Immunoregulatory Role of the PD1 Pathway in Juvenile Idiopathic Arthritis.

To investigate the immunoregulatory role of the Programmed-cell-Death-protein-1 (PD1) pathway, an inhibitory immune checkpoint, in Juvenile Idiopathic Arthritis (JIA). The PD1 expression on CD4+ and CD8+ T-cells was determined by flow cytometry and the PD1 soluble form (sPD1) levels by ELISA, in peripheral blood (PB)/serum and synovial fluid (SF) samples of JIA patients and healthy controls (HCs). We searched for any association in-between the biomarkers and with JIA activity. 101 Caucasian patients (69 female), aged 12 (8-15) years, and 20 HCs participated in this study. The PB PD1 expression on T-cells was higher in: a. JIA patients vs HCs (CD4: 1.24% vs 0.32%, p=0.007, CD8: 1.6% vs 0.4%, p=0.002). b. active vs inactive JIA (CD4: 1.44% vs 0.87%, p=0.072, CD8: 2.1% vs 0.93%, p=0.005). The SF PD1 expression on T-cells correlated strongly and positively with the disease activity (CD4: ρ=0.55, p=0.022, CD8: ρ=0.555, p=0.026). The SF PD1 expression on CD8 T-cells was higher in patients on-treatment vs those off-treatment (21.3% vs 5.83% p=0.004). The sPD1 levels were higher in the SF vs the serum (801pg/ml vs 367.2, p=0.013), without an association with disease activity. These results indicate an up-regulation of the PD1-pathway in JIA, at least quantitatively, especially in active disease. sPD1 is compartmentally produced at the inflamed joints. Further investigation in a larger sample of JIA patients may verify these observations and contribute to unravelling the precise role of the PD1 pathway in the pathogenesis and persistence of the joint inflammation.

Duplex Vertical-Flow Rapid Tests for Point-of-Care Detection of Anti-dsDNA and Anti-Nuclear Autoantibodies.

The goal of this study is to develop a rapid diagnostic test for rheumatic disease and systemic lupus erythematosus (SLE) screening. A novel rapid vertical flow assay (VFA) was engineered and used to assay anti-nuclear (ANA) and anti-dsDNA (αDNA) autoantibodies from systemic lupus erythematosus (SLE) patients and healthy controls (HCs). Observer scores and absolute signal intensities from the VFA were validated via ELISA. The rapid point-of-care VFA test that was engineered demonstrated a limit of detection of 0.5 IU/mL for ANA and αDNA autoantibodies in human plasma with an inter-operator CV of 19% for ANA and 12% for αDNA. Storage stability was verified over a three-month period. When testing anti-dsDNA and ANA levels in SLE and HC serum samples, the duplex VFA revealed 95% sensitivity, 72% specificity and an 84% ROC AUC value in discriminating disease groups, comparable to the gold standard, ELISA. The rapid αDNA/ANA duplex VFA can potentially be used in primary care clinics for evaluating patients or at-risk subjects for rheumatic diseases and for planning follow-up testing. Given its low cost, ease, and rapid turnaround, it can also be used to assess SLE prevalence estimates.

Comparative untargeted metabolomics analysis of serum metabolic alterations in patients infected with hepatitis B virus genotypes B and C

The clinicopathological characteristics and outcomes of patients infected with hepatitis B virus (HBV) differ between genotypes B and C. However, the potential metabolic mechanisms and differences in certain biological pathways associated with different HBV genotypes remain unclear to date. To obtain a better understanding of the effects of liver diseases caused by HBV genotypes B and C on host metabolism, an untargeted metabolomics analysis was performed to assess the differences among the serum metabolic profiles of healthy controls and patients infected with genotypes B or C. Here, a total of 54 serum samples were obtained from healthy controls and patients with chronic HBV infections caused by genotypes B and C, and the samples were subjected to metabolomics analysis. The serum metabolite profiles were analyzed using an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UHPLC-Q-TOF/MS)-based untargeted metabolomics approach for identifying the differentially expressed metabolites that were upregulated or downregulated by at least 1.5-foldin the serum samples of patients infected with HBV genotypes B and C. A total of 63 metabolites were found to be differentially expressed between the serum samples of healthy controls and patients infected with HBV genotype B, while 57 metabolites were differentially expressed between the serum samples of healthy controls and patients infected with HBV genotype C. The majority of these metabolites were involved in regulating the bile acid (BA) metabolism pathways. The results also indicated that 8 differentially expressed metabolites can serve as potential metabolic biomarkers for distinguishing between individuals infected with the HBV genotype B and C. Altogether, the untargeted metabolomics analysis revealed that infection with HBV genotypes B and C is associated with different metabolic profiles, and the study provided fundamental information for further detailed investigations of the mechanism underlying metabolic dysregulation in HBV infections.

Diagnostic value of anti-integrin αvβ6 antibodies in ulcerative colitis

Ulcerative colitis (UC)-related mucosal inflammation is characterized by the production of various autoantibodies with limited clinical relevance. Recent studies have shown that circulating levels of IgG against integrin αvβ6 are increased in UC patients as compared to Crohn's disease (CD) patients and healthy controls (HC). The present study assessed the diagnostic value of circulating IgG anti-αvβ6 in UC.Sera were prospectively collected from 108 outpatients with UC, 103 patients with CD, and 62 HC, and the levels of IgG anti-αvβ6 were measured using a commercially available ELISA kit. The cut-off for positive results was defined as the 95th percentile of the values of the autoantibodies in HC serum samples. Levels of IgG anti-αvβ6 were significantly higher in UC than in CD patients, including those with colonic localization, and HC. Fifty-six of the 108 (51.8%) UC patients had a positive test whereas only 17/103 (16.5%) patients with CD, and among these, 4/16 (25%) patients with colonic CD, were positive. In UC, there was no statistical difference between patients with IgG anti-αvβ6 positivity and those negative in terms of clinical disease activity, fecal calprotectin values, and disease extent. The sensitivity, specificity, predictive positive value, and predictive negative value of the test to differentiate between UC and CD were 51.9% (C.I.42.4–61.3), 83.5% (C.I. 76.3–90.7), 76.7% (C.I. 67.0–86.4), and 62.3% (C.I. 54.2–70.4) respectively.Our study confirms that anti-αvβ6 antibodies are demonstrable in the serum of the majority of UC patients and suggests the necessity of further research to understand if the anti-αvβ6 antibody determination could have a place in the clinical decision-making of IBD.

Surface-enhanced Raman spectroscopy for the characterization of different anatomical subtypes of oral cavity cancer

Background The prognosis for oral cancer patients is still very poor worldwide. Early detection and treatment therapy remain the key issue to be addressed for improved patient survival. The characteristic Raman spectral features associated with the biochemical changes in the blood serum samples can be used for the diagnosis of diseases, particularly for oral cancer. Surface-enhanced Raman spectroscopy (SERS) is a promising technique for non-invasive and early detection of oral cancer by analyzing molecular changes in body fluids.Objectives To detect oral cavity anatomical subsites (buccal mucosa, cheek, hard palate, lips, mandible, maxilla, tongue and tonsillar region) cancers by using blood serum samples, SERS with principal component analysis is used.Material and Method SERS is employed with silver nanoparticles for the analysis and detection of oral cancer serum samples by comparing with healthy serum samples. SERS spectra are recorded by Raman instrument and preprocessed using the statistical tool. Principal component analysis (PCA) and Partial least square discriminant analysis (PLS-DA) are used to discriminate between oral cancer serum samples and control serum samples.Results Some major SERS peaks are observed at 1136 cm−1 (Phospholipids) and 1006 cm−1 (Phenylalanine) remain higher in intensities for oral cancer spectra as compared to healthy spectra. The peak at 1241 cm−1 (amide III) is observed only in oral cancer serum samples while absent in healthy serum samples. Higher protein and DNA contents were detected in SERS mean spectra of oral cancer. Moreover, PCA is used to identify the biochemical differences in the form of SERS features which is used to differentiate between oral cancer and healthy blood serum samples, while PLS-DA is used to build differentiation model of oral cancer serum samples and healthy control serum samples. PLS-DA provides successful differentiation with 94% specificity and 95.5% sensitivity.Conclusions SERS can be used for the diagnosis of oral cancer and to identify metabolic changes that occur during disease development.

Correlation of serum-soluble mesothelin-related protein, HMGB1 and CA125 in diffuse malignant peritoneal mesothelioma and their combined measurement for prognosis

Diagnosis of diffuse malignant peritoneal mesothelioma (DMPM) is challenging due to the lack of efficient biomarkers for early-stage DMPM. This study was designed to characterize three serum-soluble mesothelium-related proteins, including soluble mesothelin-related protein (SMRP), high mobility group box 1 (HMGB1), and cancer antigen 125 (CA125) in diagnosing DMPM. Serum samples of DMPM patients and healthy controls were collected and an enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of HMGB1, CA125, and SMRP. Correlations between these three serum proteins were examined and the diagnostic values of the biomarkers were assessed by receiver operating curve (ROC) analysis. The combined expression levels of the three markers were also analyzed in terms of predicting patient survival. Higher levels of CA125, SMRP, and HMGB1 was found to be a prominent characteristic of DMPM patients (with > two-fold higher for all levels in DMPM patients compared to control patients, all p < .001), particularly for those with higher-stage DMPM (stage III-IV) compared with lower-stage DMPM (stage I-II) (all p < .05). HMGB1, CA125, and SMRP were all significantly inter-correlated with each other (all p < .05), the combination of the three serum markers had high sensitivity and specificity for diagnosing DMPM. Combined values of the three markers demonstrated a high AUC of 0.85, sensitivity of 78.95%, specificity of 82.75% for identifying DMPM. The combined level of the three markers also demonstrated a significant positive correlation with poor survival of DMPM patients (p = .022). CA125, SMRP, and HMGB1 are potentially valuable diagnostic biomarkers to facilitate the diagnosis of DMPM.

Comparison of Serum Metabolomics Pathways and Patterns between Patients with Major Depressive Disorder with and without Type 2 Diabetes Mellitus: An Exploratory Study.

A close relationship exists between major depressive disorder (MDD) and diabetes mellitus. The metabolomic difference and similarity between patients with and without diabetes mellitus have not been well studied in the context of MDD. We aimed to examine these differences and common serum metabolomics patterns, pathways and biomarkers that can comprehensively reflect the pathogenetic difference and similarity between these MDD groups. We performed a metabolomics analysis of serum samples of healthy controls (n = 6), patients with MDD and type 2 diabetes mellitus (n = 13), and patients with MDD without type 2 diabetes mellitus (n = 27). Metabolomics analysis was conducted using capillary electrophoresis Fourier transform mass spectrometry and a candidate compound was assigned to the 496 (290 cation, 206 anion) peaks. Moreover, we evaluated the sensitivity and specificity of the candidate biomarkers for distinguishing between MDD patients with or without type 2 diabetes mellitus. Principal component analysis revealed no clear distinction among the three groups, while naive partial least squares discriminant analysis yielded three relatively good and distinct populations based on the first principal component. Energy conversion by the tricarboxylic acid cycle represented the highest percentage among the top 30 positive factors of the first principal component, and glutamate metabolism and urea cycle represented the highest percentage among the top 30 negative factors of the first principal component. Synthesis and degradation of ketone bodies had high impact in MDD with type 2 diabetes mellitus group and taurine and hypotaurine metabolism had high impact in MDD without type 2 diabetes mellitus group for the pathway. Patterns of serum metabolites may be different among MDD with type 2 diabetes mellitus, MDD without type 2 diabetes mellitus, and healthy controls groups. Specifically, comorbid type 2 diabetes mellitus could affect metabolomics pathway and alter the distribution of serum metabolites in patients with MDD. These findings may shed light on the influence of the type 2 diabetes on the pathophysiology of MDD.

Screening and Analysis of Serum Protein Biomarkers Infected by Coronavirus Disease 2019 (COVID-19).

Coronavirus disease 2019 (COVID-19) has spread widely around the world, and in-depth research on COVID-19 is necessary for biomarkers and target drug discovery. This analysis collected serum from six COVID-19-infected patients and six healthy people. The protein changes in the infected and healthy control serum samples were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-performance liquid chromatography (HPLC). The differential protein signature in both groups was retrieved and analyzed by the Kyoto Encyclopedia of Gene and Genomes (KEGG), Gene ontology, COG/KOG, protein-protein interaction, and protein domain interactions tools. We shortlisted 24 differentially expressed proteins between both groups. Ten genes were significantly up-regulated in the infection group, and fourteen genes were significantly down-regulated. The GO and KEGG pathway enrichment analysis suggested that the chromosomal part and chromosome were the most enriched items. The oxytocin signaling pathway was the most enriched item of KEGG analysis. The netrin module (non-TIMP type) was the most enriched protein domain in this study. Functional analysis of S100A9, PIGR, C4B, IL-6R, IGLV3-19, IGLV3-1, and IGLV5-45 revealed that SARS-CoV-2 was closely related to immune response.

Melatonin suppresses serum starvation-induced autophagy of ovarian granulosa cells in premature ovarian insufficiency

ObjectivesPremature ovarian insufficiency (POI) refers to the decline and cessation of ovarian functions in women under 40 years of age. Melatonin (MT) acts as a protective for the ovary. This study elucidated the role of MT in autophagy of granulosa cells (GCs) in POI via modulating the phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway.MethodsThe expression levels of microRNA (miR)-15a-5p, signal transducer and activator of transcription 3 (Stat3), and relevant hormones in the clinically collected serum samples of POI patients and healthy controls were examined. Human ovarian granulosa-like tumor cells (KGN) underwent serum starvation (SS) treatment to induce POI cell models and then received MT treatment. The expression levels of miR-15a-5p, Stat3, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR in KGN cells were tested via quantitative real-time polymerase chain reaction and Western blotting. KGN cell viability was assessed by MTT assay and the protein levels of autophagy-related markers Beclin-1, microtubule-associated protein light chain 3 II/I, and p62 were detected by Western blotting. The binding relation between miR-15a-5p and Stat3 was verified via the dual-luciferase reporter gene assay. Functional rescue experiments were performed to probe the underlying role of miR-15a-5p/Stat3/the PI3K-Akt-mTOR pathway in KGN cell autophagy.ResultsmiR-15a-5p was increased whilst Stat3 was decreased in the serum of POI patients and SS-induced KGN cells. MT inhibited miR-15a-5p and Stat3, activated the PI3K-Akt-mTOR pathway, and repressed cell autophagy in SS-induced KGN cells. miR-15a-5p targeted and repressed Stat3 expression. Upregulation of miR-15a-5p or downregulation of Stat3 or the PI3K-Akt-mTOR pathway promoted KGN cell autophagy.ConclusionMT suppressed miR-15a-5p and activated Stat3 and the PI3K-Akt-mTOR pathway, finally impeding SS-induced autophagy of GCs.

Comparative serum proteomic analysis of a selected protein panel in individuals with schizophrenia and bipolar disorder and the impact of genetic risk burden on serum proteomic profiles

The diagnostic criteria for schizophrenia (SCZ) and bipolar disorder (BD) are based on clinical assessments of symptoms. In this pilot study, we applied high-throughput antibody-based protein profiling to serum samples of healthy controls and individuals with SCZ and BD with the aim of identifying differentially expressed proteins in these disorders. Moreover, we explored the influence of polygenic burden for SCZ and BD on the serum levels of these proteins. Serum samples from 113 individuals with SCZ and 125 with BD from the PsyCourse Study and from 44 healthy controls were analyzed by using a set of 155 antibodies in an antibody-based assay targeting a selected panel of 95 proteins. For the cases, genotyping and imputation were conducted for DNA samples and SCZ and BD polygenic risk scores (PRS) were calculated. Univariate linear and logistic models were used for association analyses. The comparison between SCZ and BD revealed two serum proteins that were significantly elevated in BD after multiple testing adjustment: “complement C9” and “Interleukin 1 Receptor Accessory Protein”. Moreover, the first principal component of variance in the proteomics dataset differed significantly between SCZ and BD. After multiple testing correction, SCZ-PRS, BD-PRS, and SCZ-vs-BD–PRS were not significantly associated with the levels of the individual proteins or the values of the proteome principal components indicating no detectable genetic effects. Overall, our findings contribute to the evidence suggesting that the analysis of circulating proteins could lead to the identification of distinctive biomarkers for SCZ and BD. Our investigation warrants replication in large-scale studies to confirm these findings.

Antineuronal Autoantibodies in the Cerebrospinal Fluid and Serum From 106 Patients With Recent-Onset Depression Compared With 106 Individually Matched Healthy Control Subjects

No large studies have investigated the prevalence of cerebrospinal fluid antineuronal autoantibodies in isolated depression. In this case-control study comparing 106 patients with isolated depression (ICD-10 code F32) with 106 healthy control subjects, cerebrospinal fluid and serum samples were tested for 7 immunoglobulin G autoantibodies using commercial fixed cell-based assays. To explore validity of methods, positive samples were retested twice by cell-based assays and once by tissue-based assays (monkey cerebellum). The prevalence of any of the antineuronal autoantibodies in cerebrospinal fluid was 0.0% in both groups and the seroprevalence was 0.9% in both groups, based on consistent findings in cell-based assays. However, all samples were negative by the tissue-based assay. Evaluation of antineuronal autoantibodies in cerebrospinal fluid cannot be recommended routinely for patients with isolated depression of moderate severity. Future studies of isolated depression should consider much larger sample sizes and evaluation of antineuronal autoantibodies using modalities other than commercial kits.

Lithocholic acid promotes rosacea-like skin inflammation via G protein-coupled bile acid receptor 1

BackgroundRosacea is a chronic inflammatory skin disorder with unclear etiology. Evidence showed that immunoinflammatory dysregulation was involved in the pathogenesis. Bile acids, as important participants of hepatoenteric circulation, play a vital role in immunoinflammatory regulation through peripheral blood circulation. However, whether it has effects on rosacea remains unknown. MethodsHere, we performed a bile acid analysis on the serum samples of rosacea patients and healthy controls. Then we gavage G protein-coupled bile acid receptor 1 (TGR5) knockout mice with lithocholic acid (LCA) based on a LL37-induced rosacea-like model. We further overexpress TGR5 in HaCaT keratinocytes to figure out the downstream pathway. ResultsWe found varied bile acid profile in the peripheral blood circulation of patients, especially the most significant increase in LCA. LCA promoted skin inflammation in LL37-induced rosacea-like mouse model. Our in vivo and in vitro results further demonstrated that LCA induced inflammatory cytokines and chemokines, thus exacerbated rosacea-like skin inflammation, via TGR5 in keratinocytes and LL37-induced rosacea-like mouse model. ConclusionsTherefore, we conclude that LCA promotes skin inflammation of rosacea via TGR5, and LCA-TGR5 axis may be a novel therapeutic target for rosacea.

Potential Serum Biomarkers Associated with Premature Rupture of Fetal Membranes in the First Trimester.

Premature rupture of the fetal membranes (PROM) is a common and important obstetric complication with increased risk of adverse consequences for both mothers and fetuses. An accurate and timely method to predict the occurrence of PROM is needed for ensuring maternal and fetal safety. Untargeted metabolomics was applied to characterize metabolite profiles related to PROM in early pregnancy. 41 serum samples from pregnant women who developed PROM later in gestation and 106 from healthy pregnant women as a control group, were analyzed. Logistic regression analysis was adjusted to analyze a PROM prediction model in the first trimester. A WISH amniotic cell viability assay was applied to explore the underlying mechanisms involved in PROM, mediated by C8-dihydroceramide used to mimic a potential biomarker (Cer 40:0; O2). Compared with healthy controls, 13 serum metabolites were identified. The prediction model comprising four compounds (Cer 40:0; O2, sphingosine, isohexanal and PC O-38:4) had moderate accuracy to predict PROM events with the maximum area under the curve of a receiver operating characteristics curve of approximately 0.70. Of these four compounds, Cer 40:0; O2 with an 1.81-fold change between PROM and healthy control serum samples was defined as a potential biomarker and inhibited the viability of WISH cells. This study sheds light on predicting PROM in early pregnancy and on understanding the underlying mechanism of PROM. Trial Registration: This study protocol has been registered at www.ClinicalTrials.gov, CT03651934, on 29 August 2018 (prior to recruitment).

Blood Eosinophils Are Associated with Efficacy of Targeted Therapy in Patients with Advanced Melanoma.

Simple SummaryDespite the advances in treatment of patients diagnosed with advanced melanoma, long-term benefits remain limited due to primary and acquired resistance. Reliable biomarkers may support treatment decisions and should optimize treatment efficacy. By using a homogeneous population of melanoma patients, peripheral blood eosinophils, along with their cytotoxic potential and soluble markers, were evaluated for their suitability as biomarkers in patients receiving targeted therapy. High relative eosinophil (REC) counts correlated with the response to targeted therapy. In vitro experiments underlined these results showing high cytotoxicity of eosinophils towards melanoma cells, which was significantly enhanced by the addition of using targeted therapy agents. We also provide evidence of a bidirectional relationship between eosinophils and melanoma cells, which might further improve the treatment of advanced melanoma.Background: Eosinophils appear to contribute to the efficacy of immunotherapy and their frequency was suggested as a predictive biomarker. Whether this observation could be transferred to patients treated with targeted therapy remains unknown. Methods: Blood and serum samples of healthy controls and 216 patients with advanced melanoma were prospectively and retrospectively collected. Freshly isolated eosinophils were phenotypically characterized by flow cytometry and co-cultured in vitro with melanoma cells to assess cytotoxicity. Soluble serum markers and peripheral blood counts were used for correlative studies. Results: Eosinophil-mediated cytotoxicity towards melanoma cells, as well as phenotypic characteristics, were similar when comparing healthy donors and patients. However, high relative pre-treatment eosinophil counts were significantly associated with response to MAPKi (p = 0.013). Eosinophil-mediated cytotoxicity towards melanoma cells is dose-dependent and requires proximity of eosinophils and their target in vitro. Treatment with targeted therapy in the presence of eosinophils results in an additive tumoricidal effect. Additionally, melanoma cells affected eosinophil phenotype upon co-culture. Conclusion: High pre-treatment eosinophil counts in advanced melanoma patients were associated with a significantly improved response to MAPKi. Functionally, eosinophils show potent cytotoxicity towards melanoma cells, which can be reinforced by MAPKi. Further studies are needed to unravel the molecular mechanisms of our observations.

Long non-coding RNA KCNQ1OT1 overexpression promotes osteogenic differentiation of staphylococcus aureus-infected human bone mesenchymal stem cells by sponging microRNA miR-29b-3p

ABSTRACT Osteomyelitis (OM) is an orthopedic disease caused by bone infections in the bone cortex, bone marrow, periosteum, and surrounding soft tissues. Recent studies have implicated non-coding RNAs (ncRNAs) in the development of OM. However, little is known about the role of ncRNAs in the osteogenic differentiation during bone infection. In the present study, we investigated the role of KCNQ1OT1/miR-29b-3p axis in osteogenic differentiation in staphylococcus aureus (SpA)-infected human bone mesenchymal stem cells (hBMSCs). We first examined the expression of lncRNA KCNQ1OT1 and miR-29b-3p in the serum samples of OM patients and healthy controls. We also infected hBMSCs with different concentrations of SpA and studied the osteogenic differentiation after infection. Our results revealed that KCNQ1OT1 was downregulated while miR-29b-3p was upregulated in the serum samples of OM patients, as well as in SpA-infected hBMSCs. Overexpression of KCNQ1OT1 ameliorated the damage in hBMSCs caused by SpA infection. KCNQ1OT1 could support hBMSCs osteogenic differentiation by enhancing ALP activity, alizarin red S accumulation, expressions of osteogenic markers, and attenuating inflammatory responses after SpA infection. We further showed that miR-29b-3p was a downstream target of KCNQ1OT1, mediating the osteogenic differentiation of hBMSCs during SpA infection. Our data suggest that KCNQ1OT1 could ameliorate the SpA-induced suppression of osteogenic differentiation in hBMSCs by sponging miR-29b-3p. Modulating KCNQ1OT1 expression may serve as a strategy to ameliorate osteomyelitis.

Long non-coding RNA cancer susceptibility candidate 2 regulates the function of human fibroblast-like synoviocytes via the microRNA-18a-5p/B-cell translocation gene 3 signaling axis in rheumatoid arthritis

ABSTRACT Rheumatoid arthritis (RA) is a perennial inflammatory condition. Preliminary research indicated that long non-coding (lnc)RNA cancer susceptibility candidate 2 (CASC2) was downregulated in the serum of RA patients. Our study was designed to reveal the roles of lncRNA CASC2 in RA and the latent mechanisms underlying its role. Bioinformatics method (Starbase) and dual-luciferase reporter assay revealed that microRNA (miR)-18a-5p directly interacted with lncRNA CASC2. Furthermore, lncRNA CASC2 and miR-18a-5p expression in the serum samples of RA patients and healthy controls were measured via reverse transcription-quantitative PCR. Compared with the healthy subjects, lncRNA CASC2 was downregulated, whereas miR-18a-5p was upregulated in patients with RA. Overexpression of lncRNA CASC2 decreased the viability of human fibroblast-like synoviocytes (HFLSs) and induced apoptosis, as revealed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and flow cytometry analyses. Furthermore, the Western blotting assay suggested that Bax was upregulated and Bcl-2 was downregulated in lncRNA CASC2 up-regulated HFLSs. Downregulation of tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, matrix metalloproteinase (MMP)1, and MMP3 levels by lncRNA CASC2 up-regulation was determined using enzyme-linked immunosorbent assays (ELISAs). However, HFLSs co-transfected with miR-18a-5p mimic exhibited opposite effects compared with the case for the overexpression of lncRNA CASC2. The aforementioned methods were used to verify that a binding site exists between B-cell translocation gene 3 (BTG3) and miR-18a-5p. The effects of miR-18a-5p inhibitor on HFLSs were reversed by BTG3 silencing. Overall, lncRNA CASC2 alleviated RA by adjusting the miR-18a-5p/BTG3 signaling axis and could serve as a novel therapeutic option for RA.

Circulating Vimentin Over-Expression in Patients with Oral Sub Mucosal Fibrosis and Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common (90%) types of oral carcinomas in the world. It is the 2nd most common and 3rd deadliest cancer in India. The lack of early detection marker is one of the major causes of worst prognosis. The vimentin belongs to intermediate filament family proteins which plays significant role in maintaining cellular integrity. Over-expression of vimentin has been widely reported in many epithelial cancers however the information regarding its prevalence in the oral cancers still needs further scientific intervention. The expression level of circulating vimentin protein in serum samples (n = 30) of oral submucous fibrosis (OSMF), OSCC patients and healthy controls were measured by performing ELISA. The serum level of vimentin was significantly higher in OSMF (p < 0.01) and OSCC (p < 0.003) patients as compared to healthy subjects. The circulating vimentin levels showed a gradual increase with increasing disease status (normal < OSMF < OSCC). Circulatory levels of vimentin may ba useful indicator of disease progression and as a suitable target for therapeutic intervention of oral submucous fibrosis and oral carcinoma.

Differential expression of miRNAs related to autophagy pathway in tissue and serum samples of colorectal cancer patients.

This study was aimed to investigate the relationship of miR-17-5p, miR-30b, miR-30d, miR-216a and miR-216b associated with autophagy gene beclin 1, and beclin 1 gene with colorectal cancer (CRC). Forty-seven patients with CRC and 50 healthy individuals with no cancer history were included in this study. In the serum, tumor and non-tumoral tissue samples of the CRC patients, and in the serum samples of the healthy subjects, expression levels of miRNAs were detected by qRT-PCR. The beclin 1 gene expression levels were determined by qRT-PCR, and protein levels were determined by Western blot method in tumor and non-tumor tissue samples of the patients. The miR-17-5p and miR-30d expressions were found to be higher in tumor tissue as compared to patient non-tumor tissues, while expressions of beclin-1, miR-30b and miR-216a were found to be lower. In addition, the beclin-1 protein levels were significantly decreased in the tumor tissue as compared to those in the patient non-tumor tissues. The miR-30d expression was significantly reduced in the serum of the patients when the serum samples of CRC patients and healthy controls were compared. The beclin 1 gene may play a role as a tumor suppressor in CRC. Moreover, these miRNAs cannot be used as highly reliable biomarkers in serum for CRC diagnosis (Tab. 2, Fig. 6, Ref. 46).

Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics.

Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise N-glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers. Selective reaction monitoring assay using synthetic glycopeptides as calibration standards allowed “top-down glycopeptidomics” for the absolute quantitation of targeted label-free glycopeptides in a range from 313.3 to 697.5 nM in the complex tryptic digests derived from serum samples of RCC patients and healthy controls. Our results provided evidence that the Asn374 residue of human clusterin is modified dominantly with the Neu5Acα2,6Gal structure and the levels of clusterin bearing an A2G2S2 with homo Neu5Acα2,6Gal terminals at Asn374 decrease significantly in RCC patients as compared with healthy controls. The present study elicits that a new strategy integrating the bottom-up glycoproteomics with top-down glycopeptidomics using structure-defined synthetic glycopeptides enables the confident identification and quantitation of the glycopeptide targets pre-determined by the existing methods for intact glycopeptide profiling.

Virus discovery in chronic inflammatory demyelinating polyneuropathy

The events triggering and/or sustaining the auto-immune response underlying chronic inflammatory demyelinating polyneuropathy (CIDP) are unknown. Similar to Guillain-Barré syndrome (GBS), a viral infection might play a role in CIDP. In this study, an virus detection method (VIDISCA-next generation sequencing) capable of detecting known and unknown viruses, was used to analyze the virome in serum of 47 CIDP patients at different time points of the disease and, when available, in cerebrospinal fluid (CSF) samples (N: 17). Serum samples of GBS patients (N:24) and healthy controls (N:114) were used for comparisons. In 5/47 (10.6%; 95% CI: 4–23) CIDP samples, 10/24 (42%; 95% CI: 22–63) GBS samples and 32/114 (28.1%; 95% CI: 20–37) healthy controls samples, anelloviruses were detected, generally regarded as a non-pathogenic species. Parvovirus B19 and GB virus C were found in two CIDP samples (4%). Parvovirus B19, HIV-1 and GB virus C were found in three GBS samples (13%). In 2/17 CIDP CSF samples, an anellovirus and polyomavirus were detected, probably due to contamination during lumbar puncture. No sequences of other viruses were detected in serum or CSF. A (persistent) viral infection sustaining the auto-immune response in CIDP seems therefore unlikely.