Data-Independent Acquisition-Based Quantitative Proteomic Analysis Reveals Potential Salivary Biomarkers of Primary Sjögren's Syndrome.

Objective As primary Sj?gren's syndrome (pSS) primarily affects the salivary glands, saliva can serve as an indicator of the glands' pathophysiology and the disease's status. This study aims to illustrate the salivary proteomic profiles of pSS patients and identify potential candidate biomarkers for diagnosis.Methods The discovery set contained 49 samples (24 from pSS and 25 from age- and gender-matched healthy controls [HCs]) and the validation set included 25 samples (12 from pSS and 13 from HCs). Totally 36 pSS patients and 38 HCs were centrally randomized into the discovery set or to the validation set at a 2:1 ratio. Unstimulated whole saliva samples from pSS patients and HCs were analyzed using a data-independent acquisition (DIA) strategy on a 2D LC?HRMS/MS platform to reveal differential proteins. The crucial proteins were verified using DIA analysis and annotated using gene ontology (GO) and International Pharmaceutical Abstracts (IPA) analysis. A prediction model for SS was established using random forests.Results A total of 1,963 proteins were discovered, and 136 proteins exhibited differential representation in pSS patients. The bioinformatic research indicated that these proteins were primarily linked to immunological functions, metabolism, and inflammation. A panel of 19 protein biomarkers was identified by ranking order based on P-value and random forest algorichm, and was validated as the predictive biomarkers exhibiting good performance with area under the curve (AUC) of 0.817 for discovery set and 0.882 for validation set.Conclusions The candidate protein panel discovered may aid in pSS diagnosis. Salivary proteomic analysis is a promising non-invasive method for prognostic evaluation and early and precise treatments for pSS patients. DIA offers the best time efficiency and data dependability and may be a suitable option for future research on the salivary proteome.

Ideal physical activity in association with incident ankylosing spondylitis: a community-based, prospective cohort study.

OBJECTIVES: Only limited risk factors for ankylosing spondylitis (AS) have been identified to date. Therefore, we aimed to explore whether cardiovascular health (CVH) behaviours and factors are associated with the risk of developing AS. METHODS: Patients with incident AS were identified in cohorts from two ongoing prospective studies. Assessments were made of the association of AS with individual baseline cardiovascular health lifestyle behaviours (including smoking status, body mass index, physical activity and diet) and cardiovascular health factors (including total cholesterol levels, blood pressure levels and fasting plasma glucose levels), and with a cardiovascular health metric determined by the number of ideal behaviours and factors. Cox regression analysis was used for the estimation of hazard ratios (HRs) for AS. RESULTS: Among 124,303 participants, incident AS was identified in 53 individuals within the 8 years of follow-up. For participants with ideal physical activity (>80 min/week) the HR was 0.21 (95% CI 0.05-0.89) compared with participants without ideal physical activity after adjusting for potential confounders. No signi cant risk of developing AS was associated with baseline smoking, diet, body mass index, blood pressure, fasting blood glucose or total cholesterol status, nor did cardiovascular health metrics. CONCLUSIONS: Adherence to ideal physical activity may reduce the risk of developing AS.

Metagenomic profiling of the pro-inflammatory gut microbiota in ankylosing spondylitis.

OBJECTIVE: Gut dysbiosis has been reported implicated in ankylosing spondylitis (AS), a common chronic inflammatory disease mainly affects sacroiliac joints and spine. Utilizing deep sequencing on the feces of untreated AS patients, our study aimed at providing an in-depth understanding of AS gut microbiota. METHODS: We analyzed the fecal metagenome of 85 untreated AS patients and 62 healthy controls by metagenomic shotgun sequencing, and 23 post-treatment feces of those AS patients were collected for comparison. Comparative analyses among different cohorts including AS, rheumatoid arthritis and Behcet's disease were performed to uncover some common signatures related to inflammatory arthritis. Molecular mimicry of a microbial peptide was also demonstrated by ELISpot assay. RESULTS: We identified AS-enriched species including Bacteroides coprophilus, Parabacteroides distasonis, Eubacterium siraeum, Acidaminococcus fermentans and Prevotella copri. Pathway analysis revealed increased oxidative phosphorylation, lipopolysaccharide biosynthesis and glycosaminoglycan degradation in AS gut microbiota. Microbial signatures of AS gut selected by random forest model showed high distinguishing accuracy. Some common signatures related to autoimmunity, such as Bacteroides fragilis and type III secretion system (T3SS), were also found. Finally, in vitro experiments demonstrated an increased amount of IFN-γ producing cells triggered by a bacterial peptide of AS-enriched species, mimicking type II collagen. CONCLUSIONS: These findings collectively indicate that gut microbiota was perturbed in untreated AS patients with diagnostic potential, and some AS-enriched species might be triggers of autoimmunity by molecular mimicry. Additionally, different inflammatory arthritis shared some common microbial signatures.

Expression of microRNA-29b2-c cluster is positively regulated by MyoD in L6 cells.

OBJECTIVES: To evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters. METHODS: The expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c. RESULTS: The expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells. CONCLUSION: MyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Transforming growth factor-ß1 induces epithelial-to-mesenchymal transition in human lung cancer cells via PI3K/Akt and MEK/Erk1/2 signaling pathways.

Metastasis of tumor cells is associated with epithelial-to-mesenchymal transition (EMT), which is a process whereby epithelial cells lose their polarity and acquire new features of mesenchyme. EMT has been reported to be induced by transforming growth factor-ß1 (TGF-ß1), but its mechanism remains elusive. In this study, we performed a study to investigate whether PI3K/Akt and MAPK/Erk1/2 signaling pathways involved in EMT in the human lung cancer A549 cells. The results showed that after treated with TGF-ß1 for 48 h, A549 cells displayed more fibroblast-like shape, lost epithelial marker E-cadherin and increased mesenchymal markers Vimentin and Fibronectin. Moreover, TGF-ß1-induced EMT after 48 h was accompanied by increased of cell migration and change of Akt and Erk1/2 phosphorylation. In addition, EMT was reversed by PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126, which suggested that A549 cells under stimulation of TGF-ß1 undergo a switch into mesenchymal cells and PI3K/Akt and MAPK/Erk1/2 signaling pathways serve to regulate TGF-ß1-induced EMT of A549 cells.

PTEN and PDCD4 are bona fide targets of microRNA-21 in human cholangiocarcinoma.

OBJECTIVE: To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells. METHODS: The expression profile of microRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line, QBC939, was evaluated by using real-time PCR analysis. The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot, respectively. The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR, locked nucleic acid in situ hybridization (LNA-ISH), and immunohistochemistry analysis. RESULTS: Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bile duct tissues (P<0.05). The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line, QBC939, inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4. Moreover, loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells. Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue. CONCLUSION: microRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN, PDCD4 are direct effectors of microRNA-21.

[Clinical analysis of 20 cases of muscular tuberculosis].

OBJECTIVE: To explore the clinical manifestations of muscular tuberculosis (MT) and analyze its risk factors. METHODS: Twenty MT patients were recruited from our department during 2000 - 2010. There were 9 males and 11 females with an average age of 43.5 years old. And their clinical manifestations were recorded and analyzed. RESULTS: All patients had local masses. And 19 patients had the involvement of single muscle and multiple muscles were involved in 1 patient. Gastrocnemius was affected in 9 patients. Nine patients had a previous history of tuberculosis or suffered concurrent tuberculosis of other body parts. Three patients with immune system disease received glucocorticoid therapy. And 11 patients underwent PPD (purified protein derivative) test and only 1 was strongly positive while 10 others were negative. MT was confirmed by pathological examinations in 20 cases. All patients underwent muscle biopsy and received effective chemotherapy. CONCLUSION: As a kind of systemic disease, MT is mainly characterized by painful or painless muscle mass. The patients with a history of tuberculosis, tuberculosis of other body parts and immune system disease are susceptible to MT. Diagnosis is mainly made through biopsy. And chemotherapy is effective.

Transforming growth factor-ß1 promotes lung adenocarcinoma invasion and metastasis by epithelial-to-mesenchymal transition.

Lung cancer is a highly malignant carcinoma, and most deaths of lung cancer are caused by metastasis. The alterations associated with epithelial-to-mesenchymal transition (EMT) may be related to the cancer cell metastasis. Nevertheless, the mechanism of lung cancer metastasis remains unclear. We conducted a study in vitro to investigate whether transforming growth factor-ß1 (TGF-ß1) could induce changes of, such as cell morphology, expression of relative protein markers, and cellular motile and invasive activities. In this research, the changes of cell morphology were first investigated under a phase contrast microscope, then western blotting was employed to detect the expression of E-cadherin, vimentin, and fibronectin, and finally cell motility and invasion were evaluated by cell wound-healing as well as invasion assays. The data indicated that human lung adenocarcinoma cell lines, A-549 and PC-9 cells of epithelial cell characteristics, were induced to undergo EMT by TGF-ß1. Following TGF-ß1 treatment, cells showed dramatic morphological changes assessed by phase contrast microscopy, accompanied by decreased epithelial marker E-cadherin and increased mesenchymal markers vimentin and fibronectin. More importantly, cell motility and invasion were also enhanced in the EMT process. These results indicated that TGF-ß1 may promote lung adenocarcinoma invasion and metastasis via the mechanism of EMT.

[Epithelial-mesenchymal transition (EMT) and its effect on microRNA expression in lung cancer].

OBJECTIVE: To investigate the effect of epithelial-mesenchymal transition (EMT) on the expression of microRNAs (miRNAs) in lung cancer A549 cells. METHODS: Transforming growth factor beta-1 (TGF-beta 1) in different concentrations was used to induce EMT in lung cancer A549 cells. The morphological changes were observed under phase-contrast microscope. The changes of EMT-related proteins were analyzed by Western blot. The changes of miRNAs expression after EMT were detected by microRNA (miRNA) array. Real time quantitative RT-PCR was applied to verify the reliability of miRNA array results. RESULTS: The lung cancer A549 cells became elongated and the cell-cell junction became loose after EMT. The epithelial protein marker E-cadherin was down-regulated and the mesenchymal protein markers vimentin and fibronectin up-regulated. There were 51 miRNAs showing statistically significant changes of expression more than double (P<0.05) after EMT. Among them 18 were up-regulated and 33 down-regulated. Of them, mir-33a was down-regulated by 92.8% and mir-193a-3p by 86.5%. Real time quantitative RT-PCR showed that mir-33a was down-regulated by 73.1% and mir-193a-3p by 56.6%. CONCLUSION: Epithelial-mesenchymal transition has effects on the expression of miRNAs, and miRNAs may regulate the invasion and metastasis of lung cancer cells via EMT.

[The diagnostic value of anti-SSA antibody in primary Sjögren's syndrome].

OBJECTIVE: According to international classification criteria (2002) on Sjögren's syndrome, labial pathology was still considered as a major criterion for diagnosis. Standard labial biopsy was hard to be carried out in China. This study is to evaluate whether the invasive labial biopsy could be replaced by noninvasive detection of serum anti-SSA antibody. METHODS: 181 Chinese patients with the initial diagnosis of primary Sjögren's syndrome in Peking Union Medical College Hospital (PUMCH) were enrolled in Sjögren's International Collaborative Clinical Alliance (SICCA). All patients received standard labial biopsies (area of salivary gland tissue ≥ 4 mm²) and focal score (FS) of focal lymphatic sialadenitis were confirmed by pathologists from school of stomatology, University California of San Francisco (UCSF). Anti-SSA antibodies in sera of all patients were detected by double immunodiffusion (DID), Western blot in PUMCH and by enzyme-linked immunosorbent assay (ELISA) in central laboratory of SICCA. The correlation between labial pathological findings and serum anti-SSA antibody was studied by chi² test and the concordance was calculated by unweighted Kappa. RESULTS: (1) Bivariate analysis revealed strong associations of FS > 1 with the presence of anti-SSA antibody by DID (83.9% vs 42.0%, P < 0.0001). The accordance between FS and antibody detection by DID was fine with a kappa value of 0.432. However, there were 16.1% false-positive antibody reports and 42.0% false-negative antibody reports. (2) FS > 1 was strongly associated with the presence of anti-SSA antibody by Western blot (83.0% vs 51.7%, P < 0.0001). But the accordance between FS and antibody detection by Western blot was only fair with a kappa value of 0.316. There were 17.0% false-positive antibody reports and 51.7% false-negative antibody reports. (3) FS > 1 was strongly associated with the presence of anti-SSA antibody by ELISA (81.5% vs 38.6%, P < 0.0001). The accordance between FS and antibody detection by ELISA was fine with a kappa value of 0.427. There were 18.5% false-positive antibody reports and 38.6% false-negative antibody reports. CONCLUSION: In Sjögren's syndrome, labial biopsy with FS > 1 finding is strongly associated with anti-SSA antibody. Positive results of anti-SSA antibodies by DID or ELISA may indicate FS > 1, thus labial biopsy could relatively be avoided, negative results may need further standard labial biopsy procedure to confirm the diagnosis of Sjögren's syndrome.