Characterised intron retention profiles in muscle tissue of idiopathic inflammatory myopathy subtypes.

OBJECTIVES: Idiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases. Intron retention (IR) serves as an important post-transcriptional and translational regulatory mechanism. This study aims to identify changes in IR profiles in IIM subtypes, investigating their influence on proteins and their correlations with clinical features. METHODS: RNA sequencing and liquid chromatography-tandem mass spectrometry were performed on muscle tissues obtained from 174 patients with IIM and 19 controls, following QC procedures. GTFtools and iREAD software were used for IR identification. An analysis of differentially expressed IRs (DEIs), exons and proteins was carried out using edgeR or DEP. Functional analysis was performed with clusterProfiler, and SPIRON was used to assess splicing factors. RESULTS: A total of 6783 IRs located in 3111 unique genes were identified in all IIM subtypes compared with controls. IIM subtype-specific DEIs were associated with the pathogenesis of respective IIM subtypes. Splicing factors YBX1 and HSPA2 exhibited the most changes in dermatomyositis and immune-mediated necrotising myopathy. Increased IR was associated with reduced protein expression. Some of the IIM-specific DEIs were correlated with clinical parameters (skin rash, MMT-8 scores and muscle enzymes) and muscle histopathological features (myofiber necrosis, regeneration and inflammation). IRs in IFIH1 and TRIM21 were strongly correlated with anti-MDA5+ antibody, while IRs in SRP14 were associated with anti-SRP+ antibody. CONCLUSION: This study revealed distinct IRs and specific splicing factors associated with IIM subtypes, which might be contributing to the pathogenesis of IIM. We also emphasised the potential impact of IR on protein expression in IIM muscles.

Clinical application of calcium silicate-based bioceramics in endodontics.

Pulp treatment is extremely common in endodontics, with the main purpose of eliminating clinical symptoms and preserving tooth physiological function. However, the effect of dental pulp treatment is closely related to the methods and materials used in the process of treatment. Plenty of studies about calcium silicate-based bioceramics which are widely applied in various endodontic operations have been reported because of their significant biocompatibility and bioactivity. Although most of these materials have superior physical and chemical properties, the differences between them can also have an impact on the success rate of different clinical practices. Therefore, this review is focused on the applications of several common calcium silicate-based bioceramics, including Mineral trioxide aggregate (MTA), Biodentine, Bioaggregate, iRoot BP Plus in usual endodontic treatment, such as dental pulp capping, root perforation repair, regenerative endodontic procedures (REPs), apexification, root-end filling and root canal treatment (RCT). Besides, the efficacy of these bioceramics mentioned above in human trials is also compared, which aims to provide clinical guidance for their clinical application in endodontics.

Disruption of the CCDC43-FHL1 interaction triggers apoptosis in gastric cancer cells.

The coiled-coil domain-containing protein 43 (CCDC43) is essential to promote gastric cancer (GC) proliferation and invasion, while four and a half LIM domains 1 (FHL1) involves GC cells apoptosis. We attempted to address inter-relationship between CCDC43 and FHL1 in modulating GC cells growth and apoptosis. Levels of protein expression were assessed by western blot, immunofluorescence. Using EdU, plate colony formation, Matrigel invasion and animal models, we evaluated the function in vitro and in vivo. Apoptosis was evaluated by flow cytometry and Hoechst 33258 staining. Reciprocal co-immunoprecipitation (co-IP) analyses indicated that CCDC43 physically interacted with FHL1. The expression of CCDC43 was negatively correlated with FHL1. Moreover, up-regulation of CCDC43 resulted in FHL1 level decline, and the reverse is also true. CCDC43 expressed jointly with FHL1 group significantly decreases the ability of the growth, metastasis and invasion of GC cells compared with that of the CCDC43 group. Furthermore, siRNA-mediated repression of CCDC43 results in dissociation from FHL1 and causes suppression of GC cell proliferation and metastasis. CCDC43 repression mediates the stability of FHL1 protein. In addition, CCDC43 interacts with FHL1. Knockdown of CCDC43 plus FHL1 overexpression inhibits proliferation and migration and induces apoptosis of GC cells in vitro and vivo.

The TGF-ß/miR-31/CEACAM1-S axis inhibits CD4+ CD25+ Treg differentiation in systemic lupus erythematosus.

Defects causing concomitant loss of CD25 expression in regulatory T cells (Tregs) have been identified in systemic lupus erythematosus (SLE). However, the cause of this deficiency is not fully understood. Carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1), an immune co-receptor, contributes to general T-cell function and activation. Our previous study revealed that CEACAM1 expression was upregulated in peripheral blood mononuclear cells (PBMCs) from patients with SLE. However, its role remains unclear. Herein, we confirmed CEACAM1, especially CEACAM1-S, was upregulated in PBMCs from patients with SLE. CEACAM1-S over-expression inhibits CD4+ CD25+ Treg differentiation, whereas knockdown of CEACAM1 had the opposite effect in vitro. CEACAM1-S is the target of miR-31. MiR-31 mimic inhibits CEACAM1 expression and enhances CD4+ CD25+ Treg differentiation, which was reversed by CEACAM1-S over-expression. Moreover, the circulating TGF-ß level was upregulated in SLE patients and TGF-ß reduced miR-31 expression via enhancing NF-κB activity. Importantly, CEACAM1 and TGF-ß mRNA levels were downregulated, while the miR-31 level and the abundance of CD4+ CD25+ Tregs were increased in inactive patients compared with that in patients with active SLE. In addition, CEACAM1-S expression was positively correlated with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, while CD4+ CD25+ Treg abundance and miR-31 level were negatively correlated with the SLEDAI score. In conclusion, reduced activity of miR-31 by TGF-ß, via the inhibition of NF-ᴋB, acted to inhibit the differentiation of CD4+ CD25+ Tregs by directly targeting CEACAM1-S and to promote autoimmunity.

PKM2-dependent glycolysis promotes skeletal muscle cell pyroptosis by activating the NLRP3 inflammasome in dermatomyositis/polymyositis.

OBJECTIVES: Muscle cell necrosis is the most common pathological manifestation of idiopathic inflammatory myopathies. Evidence suggests that glycolysis might participate in it. However, the mechanism is unclear. This study aimed to determine the role of glycolysis in the muscle damage that occurs in DM/PM. METHODS: Mass spectrometry was performed on muscle lesions from DM/PM and control subjects. The expression levels of pyruvate kinase isozyme M2 (PKM2), the nucleotide-binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and pyroptosis-related genes in muscle tissues or plasma were determined by real-time PCR, western blot analysis, IF and ELISA. In addition, IFNγ was used to stimulate myotubes, and the relationships among PMK2 expression, NLRP3 inflammasome activation and pyroptosis were investigated. RESULTS: Mass spectrometry and bioinformatics analysis suggested that multiple glycolysis processes, the NLRP3 inflammasome and programmed cell death pathway-related proteins were dysregulated in the muscle tissues of DM/PM. PKM2 and the NLRP3 inflammasome were upregulated and positively correlated in the muscle fibres of DM/PM. Moreover, the pyroptosis-related proteins were increased in muscle tissues of DM/PM and were further increased in PM. The levels of PKM2 in muscle tissues and IL-1ß in plasma were high in patients with anti-signal recognition particle autoantibody expression. The pharmacological inhibition of PKM2 in IFNγ-stimulated myotubes attenuated NLRP3 inflammasome activation and subsequently inhibited pyroptosis. CONCLUSION: Our study revealed upregulated glycolysis in the lesioned muscle tissues of DM/PM, which activated the NLRP3 inflammasome and leaded to pyroptosis in muscle cells. The levels of PKM2 and IL-1ß were high in patients with anti-signal recognition particle autoantibody expression. These proteins might be used as new biomarkers for muscle damage.

Evaluating protein binding specificity of titanium surfaces through mass spectrometry-based proteomics.

OBJECTIVES: To evaluate whether surface characteristics of different titanium modifications may influence the composition of the salivary pellicle on each surface by analyzing the salivary proteome through mass spectrometry-based proteomics. MATERIALS AND METHODS: Titanium discs with three surfaces modifications (PT (machined titanium), SLA (sandblasted/large-grit/acid-etched), and SLActive (modified SLA)) were characterized (topography, chemistry, and energy) prior to being exposed to saliva for 2 h to form a protein pellicle. The resultant protein layer was retrieved and analyzed through mass spectrometry (nLC-ESI-MS/MS) to examine the surface specificity for protein binding, while the proteome profile of each surface was classified. RESULTS: The proteome analysis showed that the salivary pellicle composition was more complex on rough surfaces (SLA and SLActive). Although variability in protein composition was observed between surfaces, most proteins were detected on more than one surface, indicating a limited surface specificity for protein binding. Additionally, the salivary pellicle formed on the SLActive presented a larger number of proteins associated with immune response, biological adhesion, and biomineralization. CONCLUSIONS: Although topography, chemistry, and energy differed between the surfaces, they were not determinant to produce a salivary pellicle with high surface specificity. Also, we showed that several salivary proteins adsorbed on Ti surfaces are involved in biological functions important to the biointegration. CLINICAL RELEVANCE: This study sheds light on the necessity for the development of bioactive surfaces that favors the formation of a specific protein layer that can enhance tissue response to assist the biointegration of dental implants.

Neutrophil-derived exosome from systemic sclerosis inhibits the proliferation and migration of endothelial cells.

OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, inflammation, and extensive fibrosis in multiple organs. Exosomes (EXOs) are cell-derived vesicles contained various DNAs, RNAs and proteins, and play important roles in various diseases. Here, we aimed to investigate the roles of SSc EXOs in angiogenesis related mechanisms. METHODS: EXOs were isolated from plasma, cultured peripheral blood mononuclear cells (PBMCs)/neutrophil supernatants, and identified by transmission electron microscopy. The expression of S100A8/A9 was measured by real-time PCR and ELISA. Proliferation, migration and scratch assays in human dermal microvascular endothelial cells (HDMECs) were used to study the EXOs influence. RESULTS: Plasma and neutrophil EXOs from SSc patients can suppress the proliferation and migration of HDMECs. High levels of S100A8/A9 were found in SSc EXOs which derived from plasma, PBMCs and neutrophils. The expression of S100A8/A9 in neutrophil EXOs was higher than that in PBMC EXOs in SSc patients. The proliferation and migration of HDMECs were possibly inhibited by S100A8/A9 of neutrophil EXOs. CONCLUSIONS: Neutrophil EXOs from SSc patients inhibits the proliferation and migration of HDMECs, S100A8/A9 might play an important role in this process.

Global analysis of protein expression in muscle tissues of dermatomyositis/polymyosisits patients demonstrated an association between dysferlin and human leucocyte antigen A.

OBJECTIVES: DM and PM are characterized by myofibre damage with inflammatory cell infiltration due to the strong expressions of MHC class I HLA-A and monocyte chemoattractant protein-1 (MCP-1). Dysferlin (DYSF) is a transmembrane glycoprotein that anchors in the sarcolemma of myofibres. DYSF mutation is closely associated with inherited myopathies. This study aimed to determine the role of DYSF in the development of DM/PM. METHODS: Mass spectrometry was performed in muscle tissues from DM/PM patients and controls. The DYSF levels in muscle tissue, peripheral blood cells and serum were detected by Western blotting, IF, flow cytometry or ELISA. Double IF and co-immunoprecipitation were used to investigate the relationship between DYSF and HLA-A. RESULTS: Mass spectrometry and bioinformatics analysis findings suggested the dysregulated proteins in DM/PM patients participated in common biological processes and pathways, such as the generation of precursor metabolites and energy. DYSF was upregulated in the muscle tissue and serum of DM/PM patients. DYSF was mainly expressed in myofibres and co-localized with HLA-A and MCP-1. DYSF and HLA-A expressions were elevated in myocytes and endothelial cells after being stimulated by patient serum and IFN-ß. However, no direct interactions were found between DYSF and HLA-A by co-immunoprecipitation. CONCLUSION: Our study revealed the dysregulated proteins involved in common and specific biological processes in DM/PM patient samples. DYSF is upregulated and exhibits a potential role along with that of HLA-A and MCP-1 in inflammatory cell infiltration and muscle damage during the development of DM/PM.

Snail/FOXK1/Cyr61 Signaling Axis Regulates the Epithelial-Mesenchymal Transition and Metastasis in Colorectal Cancer.

BACKGROUND/AIMS: Metastasis is the primary cause of colorectal cancer (CRC)-related death. However, the molecular mechanisms underlying metastasis in CRC remain unclear. METHODS: We evaluated mRNA and protein expression levels by quantitative real-time reverse transcription PCR, western blotting, immunofluorescence, tissue microarrays, and immunohistochemistry assays. We also assessed the migration and invasion abilities of CRC cells in vitro by wound healing assays, invasion and migration assays, western blot analysis, and immunofluorescence. Tumor metastasis was evaluated in nude mice in vivo. RESULTS: A positive correlation was observed between the expression patterns of Forkhead box k1 (FOXK1) and Snail in CRC. Luciferase reporter and chromatin immunoprecipitation assays demonstrated that Snail directly bound to and activated the human FOXK1 gene promoter. Moreover, the Snail-FOXK1 axis promote epithelial mesenchymal transition (EMT)-mediated CRC cell invasion and metastasis. FOXK1 and Snail expression levels were correlated with tumor progression and served as significant predictors of overall survival in patients with CRC. Furthermore, overexpression of FOXK1 induced the EMT by upregulating the expression of cysteine-rich angiogenic inducer 61 (Cyr61). Luciferase assays showed that Cyr61 was a direct transcriptional target of FOXK1. Down regulation of Cyr61 decreased FOXK1-enhanced "CRC cell" migration, invasion, and metastasis. Additionally, FOXK1 expression was positively correlated with Cyr61 expression and was associated with poor prognosis. CONCLUSIONS: The Snail/FOXK1/Cyr61 signaling axis regulates the EMT and metastasis of CRC.

The FOXK1-CCDC43 Axis Promotes the Invasion and Metastasis of Colorectal Cancer Cells.

BACKGROUND/AIMS: The CCDC43 gene is conserved in human, rhesus monkey, mouse and zebrafish. Bioinformatics studies have demonstrated the abnormal expression of CCDC43 gene in colorectal cancer (CRC). However, the role and molecular mechanism of CCDC43 in CRC remain unknown. METHODS: The functional role of CCDC43 and FOXK1 in epithelial-mesenchymal transition (EMT) was determined using immunohistochemistry, flow cytometry, western blot, EdU incorporation, luciferase, chromatin Immunoprecipitation (ChIP) and cell invasion assays. RESULTS: The CCDC43 gene was overexpressed in human CRC. High expression of CCDC43 protein was associated with tumor progression and poor prognosis in patients with CRC. Moreover, the induction of EMT by CCDC43 occurred through TGF-ß signaling. Furthermore, a positive correlation between the expression patterns of CCDC43 and FOXK1 was observed in CRC cells. Promoter assays demonstrated that FOXK1 directly bound and activated the human CCDC43 gene promoter. In addition, CCDC43 was necessary for FOXK1- mediated EMT and metastasis in vitro and vivo. Taken together, this work identified that CCDC43 promoted EMT and was a direct transcriptional target of FOXK1 in CRC cells. CONCLUSION: FOXK1-CCDC43 axis might be helpful to develop the drugs for the treatment of CRC.

Comparison of soluble urokinase plasminogen activator receptor, soluble triggering receptor expressed on myeloid cells 1, procalcitonin and C-reactive protein in distinguishing concurrent bacterial infection from idiopathic inflammatory myopathy.

The aim of the study was to measure the diagnostic values of biomarkers of bacterial infection in idiopathic inflammatory myopathy (IIM) patients. The serum and clinical data of 82 IIM patients with/without bacterial infection were collected. Concentrations of soluble urokinase plasminogen activator receptor (suPAR), soluble triggering receptor expressed on myeloid cells 1 (sTREM-1), procalcitonin (PCT) and C-reactive protein (CRP) were measured in IIM patients and healthy controls. There were no significant differences in serum suPAR and sTREM-1 levels between healthy controls and non-infection IIM patients. Serum levels of suPAR, sTREM-1, PCT and CRP measured in this study were significantly higher in the IIM patient group with concurrent infection than in the non-infection IIM patient group (p < 0.05). The biomarker suPAR showed the highest diagnostic value with sensitivity, specificity, positive predictive value and negative predictive value of 81.6, 77.3, 75.6 and 82.9%, respectively. Combining suPAR negative and CRP negative to rule out bacterial infection in IIM patients provides a very high specificity of 97.4%. Both suPAR and CRP positive to confirm bacterial infection give the specificity of 90.9%. The inflammatory biomarkers suPAR, sTREM-1, PCT and CRP offer diagnostic accuracy in detecting bacterial infection in IIM patients. Particularly, suPAR is the most sensitive and specific biomarker to predict bacterial infection in IIM patients. Combination of suPAR and CRP serum levels provides an even better confirmation of bacterial infection.

Acute vibration induces transient expression of anabolic genes in the murine intervertebral disc.

OBJECTIVE: Low-amplitude whole-body vibration has been adopted for the treatment of back pain and spinal disorders. However, there is limited knowledge of the impact of vibration on the intervertebral disc (IVD). This study was undertaken to examine the effects of acute vibration on anabolic and catabolic pathways in the IVD and to characterize the dependence of these changes on time and frequency. METHODS: Custom-designed platforms were developed to apply acute vibration to ex vivo and in vivo mouse models. Spinal segments (ex vivo) or mice (in vivo) were subjected to vibration (for 30 minutes at 15-90 Hz with peak acceleration at 0.3g), and IVDs were examined at specific time points after vibration. Gene expression was quantified using real-time polymerase chain reaction, and protein levels were examined by quantitative mass spectrometry and immunofluorescence. RESULTS: In the ex vivo model, acute vibration at 15 Hz induced expression of anabolic genes (aggrecan, biglycan, decorin, type I collagen, and Sox9) and suppressed expression of Mmp13, with the most pronounced changes detected 6 hours following vibration. These beneficial effects were frequency dependent and were no longer evident between 45 and 90 Hz. In vivo, the effects on anabolic gene expression were even more robust and were accompanied by decreased expression of Adamts4, Adamts5, and Mmp3. Moreover, significant increases in the protein levels of aggrecan, biglycan, decorin, and type I collagen were detected in vivo. CONCLUSION: These findings demonstrate dramatic anabolic effects of acute vibration on IVD tissue, responses that are dependent on frequency. The similarity of the in vivo and ex vivo responses indicates that at least some effects of vibration are tissue autonomous.

Novel prognostic alternative splicing events in colorectal Cancer: Impact on immune infiltration and therapy response.

OBJECTIVE: This study aims to comprehensively analyze alternative splicing (AS) features in colorectal cancer (CRC) using integrative multi-omics and to elucidate their relationship with the CRC immune microenvironment. METHODS: Transcriptomic data, clinical information, and Percent Spliced In (PSI) values of AS events for CRC patients were obtained from The Cancer Genome Atlas (TCGA) and TCGA SpliceSeq databases. Differentially expressed AS events were identified. Univariate Cox analysis was used to pinpoint prognosis-related AS events. A prognostic risk model was developed and validated using multivariate Cox analysis, patient survival analysis, and the area under the receiver operating characteristic (ROC) curve (AUC). Gene Set Enrichment Analysis (GSEA), immune infiltration, immunotherapy, chemotherapy sensitivity analyses, and regulatory relationships between AS events and splicing factors (SFs) were conducted. Single-cell sequencing was used to study the distribution of key factors. siRNA and overexpression vectors were utilized to silence/overexpress BCAS1 in CRC cells and evaluate their effects on cell growth, migration, and invasion. Furthermore, the interaction between BCAS1 and ANO7 pre-mRNA was investigated using RIP-PCR. RESULTS: 82 prognosis-related AS events were identified in CRC patients. A 15-AS prognostic model was constructed, which correlated with immune cell infiltration and showed differences in immunotherapy and chemotherapy sensitivity. BCAS1 was identified as a potential regulator of the ANO7|58341|AT splicing event in CRC. Single-cell sequencing analysis revealed the distribution of BCAS1 and ANO7 in cancer stem cells. In vitro experiments demonstrated that overexpression of BCAS1 and silencing of ANO7 inhibit the proliferation, migration, and invasion of CRC cells. Moreover, BCAS1 suppresses the progression of CRC by modulating ANO7 alternative splicing. CONCLUSION: This study provides new insights into the role of alternative splicing in colorectal cancer, particularly the potential regulatory action of BCAS1 on the ANO7|58341|AT splicing event. It also identifies the impact of alternative splicing on the tumor microenvironment and potential implications for immunotherapy, highlighting its relevance for the in-depth study and treatment of CRC.

Relationship between Helicobacter pylori infection and digestive tract diseases and analysis of risk factors: a cross-sectional study based on 3867 Chinese patients.

Helicobacter pylori (H. pylori) infect nearly half of the global population, contributing to upper digestive tract diseases. This 2019 cross-sectional study included 3,867 patients undergoing esophagogastroduodenoscopy (EGD) and 2,875 undergoing both colonoscopy and EGD. Subjects were categorized into H. pylori positive and negative groups by rapid urease test (RUT). In addition to exploring the relationship between H. pylori infection and upper gastrointestinal diseases, this study further revealed that H. pylori infection was closely related to lower digestive tract diseases, including colorectal polyp (63.28%) and colorectal cancer (75.76%), as well as upper and lower gastrointestinal comorbidities, including chronic atrophic gastritis with colorectal polyp (79.85%), peptic ulcer with colorectal polyp (79.72%), gastric polyp with colorectal polyp (66.24%), and chronic atrophic gastritis with colorectal cancer (92.86%). Besides, a univariate logistic regression analysis was conducted to compare the differences between the two groups (including gender, nationality, marital status, smoking history, drinking history, living area, age, BMI, glycosylated hemoglobin, fasting blood glucose, total cholesterol, and triglyceride levels), the results identified marital status and age as independent risk factors for H. pylori infection (OR, 1.435; 95% CI, 1.042 to 1.977; OR, 1.007; 95% CI, 1.001 to 1.013). Further clarification of the correlation between the prevalence of gastrointestinal diseases and H. pylori infection will be important for H. pylori infection management strategies and the treatment and prevention of gastrointestinal diseases.

POU2F1 inhibits miR-29b1/a cluster-mediated suppression of PIK3R1 and PIK3R3 expression to regulate gastric cancer cell invasion and migration.

BACKGROUND: The transcription factor POU2F1 regulates the expression levels of microRNAs in neoplasia. However, the miR-29b1/a cluster modulated by POU2F1 in gastric cancer (GC) remains unknown. METHODS: Gene expression in GC cells was evaluated using reverse-transcription polymerase chain reaction (PCR), western blotting, immunohistochemistry, and RNA in situ hybridization. Co-immunoprecipitation was performed to evaluate protein interactions. Transwell migration and invasion assays were performed to investigate the biological behavior of GC cells. MiR-29b1/a cluster promoter analysis and luciferase activity assay for the 3'-UTR study were performed in GC cells. In vivo tumor metastasis was evaluated in nude mice. RESULTS: POU2F1 is overexpressed in GC cell lines and binds to the miR-29b1/a cluster promoter. POU2F1 is upregulated, whereas mature miR-29b-3p and miR-29a-3p are downregulated in GC tissues. POU2F1 promotes GC metastasis by inhibiting miR-29b-3p or miR-29a-3p expression in vitro and in vivo. Furthermore, PIK3R1 and/or PIK3R3 are direct targets of miR-29b-3p and/or miR-29a-3p, and the ectopic expression of PIK3R1 or PIK3R3 reverses the suppressive effect of mature miR-29b-3p and/or miR-29a-3p on GC cell metastasis and invasion. Additionally, the interaction of PIK3R1 with PIK3R3 promotes migration and invasion, and miR-29b-3p, miR-29a-3p, PIK3R1, and PIK3R3 regulate migration and invasion via the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway in GC cells. In addition, POU2F1, PIK3R1, and PIK3R3 expression levels negatively correlated with miR-29b-3p and miR-29a-3p expression levels in GC tissue samples. CONCLUSIONS: The POU2F1-miR-29b-3p/miR-29a-3p-PIK3R1/PIK3R1 signaling axis regulates tumor progression and may be a promising therapeutic target for GC.

Proteome and peptidome of human acquired enamel pellicle on deciduous teeth.

Understanding the composition and structure of the acquired enamel pellicle (AEP) has been a major goal in oral biology. Our lab has conducted studies on the composition of AEP formed on permanent enamel. The exhaustive exploration has provided a comprehensive identification of more than 100 proteins from AEP formed on permanent enamel. The AEP formed on deciduous enamel has not been subjected to the same biochemical characterization scrutiny as that of permanent enamel, despite the fact that deciduous enamel is structurally different from permanent enamel. We hypothesized that the AEP proteome and peptidome formed on deciduous enamel may also be composed of unique proteins, some of which may not be common with AEP of permanent enamel explored previously. Pellicle material was collected from 10 children (aged 18-54 months) and subjected to mass spectrometry analysis. A total of 76 pellicle proteins were identified from the deciduous pellicle proteome. In addition, 38 natural occurring AEP peptides were identified from 10 proteins, suggesting that primary AEP proteome/peptidome presents a unique proteome composition. This is the first study to provide a comprehensive investigation of in vivo AEP formed on deciduous enamel.

The ATF2/miR-3913-5p/CREB5 axis is involved in the cell proliferation and metastasis of colorectal cancer.

Various miRNAs have been shown to participate in the tumor progression and development of colorectal cancer (CRC). However, the role of miR-3913-5p in CRC are yet to be clearly defined. In the present study, we determine that miR-3913-5p is downregulated in CRC cell lines and CRC tissues. Exogenous miR-3913-5p expression weakens the CRC cells growth, migration and invasion. Mechanistically, miR-3913-5p directly targets the 3'UTR of CREB5. Overexpression of CREB5 reverses the suppression of CRC cells proliferation, migration and invasion induced by miR-3913-5p. Furthermore, ATF2 negatively regulates the transcription of miR-3913-5p by binding to its promoter. CREB5 can cooperate with ATF2. CREB5 is required for ATF2 in regulating miR-3913-5p. Finally, inverse correlations can be found between the expressions of miR-3913-5p and CREB5 or ATF2 in CRC tissues. Thus, a plausible mechanism of ATF2/miR-3913-5p/CREB5 axis regulating CRC progression is elucidated. Our findings suggest that miR-3913-5p functions as a tumor suppressor in CRC. ATF2/miR-3913-5p/CREB5 axis might be a potential therapeutic target against CRC progression.

Identification and characterization of histatin 1 salivary complexes by using mass spectrometry.

With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. We investigated interactions among and functions of histatin 1 and the other proteins that are present in saliva by using high-throughput mass spectrometric techniques. This led to the identification of 43 proteins able to interact with histatin 1. In addition, we found that these protein-protein interactions protect complex partners from proteolysis and modulate their antifungal activity. Our data contribute significantly to characterization of the salivary interactome and to understanding the biology of salivary protein complexes.

Engineered Salivary Peptides Reduce Enamel Demineralization Provoked by Cariogenic S. mutans Biofilm.

Engineering of the acquired enamel pellicle using salivary peptides has been shown to be a promising anticaries strategy. However, the mechanisms by which these peptides protect teeth against tooth decay are not fully understood. In this study, we evaluated the effect of the engineered salivary peptides DR9-DR9 and DR9-RR14 on enamel demineralization in two experimental conditions: (1) adsorbed onto the enamel surface forming the AEP, and (2) forming the AEP combined with their use to treat the biofilms 2×/day, using a validated cariogenic Streptococcus mutans in vitro biofilm model. Biofilms were grown for 144 h on enamel slabs and then collected to determine the bacterial viability (CFU/biofilm) and biofilm mass (mg protein/biofilm), and to extract cellular/extracellular proteins, which were characterized by mass spectrometry. The culture medium was changed 2×/day to fresh medium, and pH (indicator of biofilm acidogenicity) and calcium concentration (indicator of demineralization) was determined in used medium. DR9-RR14 peptide significantly reduced enamel demineralization (p < 0.0001) in both experimental conditions. However, this peptide did not have a significant effect on biofilm biomass (p > 0.05) nor did it modulate the expression of cellular and extracellular bacterial proteins involved in biofilm cariogenicity. These findings suggest that DR9-RR14 may control caries development mainly by a physicochemical mechanism.

Modulation of Streptococcus mutans Adherence to Hydroxyapatite by Engineered Salivary Peptides.

Since the modification of the proteinaceous components of the Acquired Enamel Pellicle (AEP) could influence the adhesion of Streptococcus mutans, the most cariogenic bacteria, to dental surfaces, we assessed if engineered salivary peptides would affect the adherence and modulate the bacterial proteome upon adherence. Single-component AEPs were formed onto hydroxyapatite (HAp) discs by incubating them with statherin, histatin-3, DR9, DR9-DR9, DR9-RR14, RR14, and parotid saliva. Then, the discs were inoculated with S. mutans UA159 and the bacteria were allowed to adhere for 2 h, 4 h, and 8 h (n = 12/treatment/time point). The number of bacteria adhered to the HAp discs was determined at each time point and analyzed by two-way ANOVA and Bonferroni tests. Cell-wall proteins were extracted from adhered, planktonic, and inoculum (baseline) bacteria and proteome profiles were obtained after a bottom-up proteomics approach. The number of adhered bacteria significantly increased over time, being the mean values obtained at 8 h, from highest to lowest, as follows: DR9-RR14 > statherin > RR14 = DR9-DR9 > DR9 = histatin3 > saliva (p < 0.05). Treatments modulated the bacterial proteome upon adherence. The findings suggested a potential use of our engineered peptide DR9-DR9 to control S. mutans biofilm development by reducing bacterial colonization.