Fabrication of multi-functional gelatin/deep eutectic solvent/polyacrylonitrile nanofiber membranes via electrospinning.

In this work, gelatin was dissolved in sodium acetate trihydrate/urea deep eutectic solvent (DES) and then mixed with polyacrylonitrile (PAN) spinning solution to prepare composite nanofiber membranes via electrospinning. The rheological properties of gelatin/DES (Gel/DES) solution and gelatin/DES/PAN (GDES/PAN) spinning solution were investigated. Besides, the water vapor transmittance rate, water contact angle, and antistatic and mechanical properties of GDES/PAN nanofiber membranes had also been evaluated and the composition and structure of GDES/PAN nanofiber membranes had also been characterized by SEM, EDS and FTIR further. Due to the introduction of DES components and gelatin, the composite nanofibers presented a smooth surface, small diameter, uniform distribution and good continuity. GDES/PAN nanofiber membranes also exhibited desirable breathable properties, hydrophilicity, antistatic properties and mechanical strength compared with the PAN nanofiber membrane. GDES/PAN nanofiber membranes would provide new opportunities for the application of gelatin and DES in the electrospinning field.

Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG via DNA methylation.

OBJECTIVE: This study aimed to investigate how mechanical force affects the proliferation of human periodontal ligament stem cells (hPDLSCs). METHODS: CCK-8 assays and staining of ki67 were performed to evaluate hPDLSCs proliferation. qRT-PCR, ELISA, or Western blot analysis were used to measure the expression levels of interleukin (IL)-6, miR-31 host gene (MIR31HG), DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3B (DNMT3B). Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) assays were conducted to determine whether MIR31HG was targeted by DNMT1 and DNMT3B. MassARRAY mass spectrometry was used to quantify DNA methylation levels of the MIR31HG promoter. RESULTS: Mechanical force inhibited hPDLSCs proliferation with the downregulation of MIR31HG and upregulation of IL-6, DNMT1 and DNMT3B. Knockdown of MIR31HG suppressed hPDLSCs proliferation, and knockdown of DNMT1 or DNMT3B reversed mechanical force-induced downregulation of MIR31HG. Dual-luciferase and ChIP assays revealed DNMT1 and DNMT3B bound MIR31HG promoter in the region 1,015 bp upstream of the transcriptional start site. Treatment with 5'-aca-2'-deoxycytidine downregulated DNA methylation level in MIR31HG gene promoter, while mechanical force promoted the methylation of MIR31HG gene promoter. CONCLUSIONS: These findings elucidated how mechanical force affects proliferation via MIR31HG in hPDLSCs, providing clues for possible MIR31HG-based orthodontic therapeutic approaches.

Genomic DNA Methylation in Diabetic Chronic Complications in Patients With Type 2 Diabetes Mellitus.

Aim: To explore the relationship between genomic DNA methylation and diabetic chronic complications. Methods: 299 patients with type 2 diabetes mellitus (T2DM) hospitalized in the Second Affiliated Hospital of Soochow University were enrolled. We divided the patients into different complications groups and corresponding non-complication groups. Clinical and biochemical parameters were compared between the two groups. The level of genomic DNA methylation in leukocytes was determined by high-performance liquid chromatography-tandem mass spectrometry. Results: (1) Age, duration of diabetes, creatinine (Cr), blood urea nitrogen (BUN), genomic DNA methylation, 24- hour urine total protein (24-hUTP), and intima-media thickness (IMT) were significantly higher in the carotid plaque (CP) group. Waist-to-hip ratio (WHR), body mass index (BMI), estimated glomerular- filtration rate (eGFR), and albumin (Alb) were significantly lower in the CP group. Gender, age and BMI were the influencing factors of CP. (2) Age, duration, Cr, BUN, urinary microalbumin creatinine ratio (UACR), systolic blood pressure (SBP), TCSS, and 24- hUTP were significantly higher in the diabetic retinopathy (DR) group. eGFR, 2h postprandial C- peptide, and Alb were lower in the DR group. Age, duration, Cr, Alb, SBP, and the presence of DN were the influencing factors of DR. (3) Age, duration, HbA1c, BUN, TCSS, SBP, and IMT(R) were significantly higher in the diabetic nephropathy (DN) group. 2h postprandial C-peptide, and Alb were lower in the DN group. HbA1c, BUN, DR, and HBP were the influencing factors of DN. (4) Age, duration, total cholesterol (TC), low-density lipoprotein (LDL-C), triglyceride (TG), Cr, BUN, uric acid (UA), and SBP were significantly higher in the diabetic peripheral neuropathy (DPN) group. The level of genomic DNA methylation and eGFR were significantly lower in the DPN group. Age, duration, LDL-C, UA, the presence of DR, and the genomic DNA methylation level were the influencing factors for DPN. Incorporating the level of genomic DNA methylation into the prediction model could improve the ability to predict DPN on the basis of conventional risk factors. Conclusion: Low level of genomic DNA methylation is a relatively specific risk factor for DPN in patients with T2DM and not a contributing factor to the other chronic complications.

An analysis of the dynamic changes in the self-efficacy and quality of life of elderly patients with chronic obstructive pulmonary disease following community-based rehabilitation.

OBJECTIVE: This study aimed to investigate the effects of community-based rehabilitation (CBR) on the self-efficacy and quality of life in elderly patients with chronic obstructive pulmonary disease (COPD). METHOD: Eighty-one elderly patients with COPD admitted to our hospital were recruited as the study cohort and were randomly divided into a control group (n=41) and a study group (n=40). The control group underwent outpatient rehabilitative treatment, and the study group additionally underwent CBR. The treatment effects of the two groups at 1 month, 3 months, and 6 months of intervention were assessed using their pulmonary function and quality of life scores. RESULTS: After completion of the CBR, the patients in the study group and the control group were scored using the CSES scale, which did not differ at 1 month of intervention, but the scores were higher in the study group than they were in the control group at 3 and 6 months of intervention (P < 0.05). The patients in the study group also scored higher on the WHOQOL-BREF scale than the control group (P < 0.05). CONCLUSION: CBR improves the self-efficacy and quality of life in elderly patients with COPD.

Value of shear wave elastography combined with the Toronto clinical scoring system in diagnosis of diabetic peripheral neuropathy.

ABSTRACT: To evaluate the diagnostic values of shear wave elastography (SWE) alone and in combination with the Toronto clinical scoring system (TCSS) on diabetic peripheral neuropathy (DPN) in patients with type 2 diabetes mellitus (T2DM).The study included 41 DPN patients, 42 non-DPN patients, and 21 healthy volunteers. Conventional ultrasonography and SWE were performed on the 2 sides of the tibial nerves, and cross-sectional area (CSA) and nerve stiffness were measured. TCSS was applied to all patients. A receiver operating characteristic curve analysis was performed.The stiffness of the tibial nerve, as measured as mean, minimum or maximum elasticity, was significantly higher in patients in the DPN group than the other groups (P < .05). The tibial nerve of subjects in the non-DPN group was significantly stiffer compared to the control group (P < .05). There was no significant difference of the tibial nerve CSA among the 3 groups (P > .05). Mean elasticity of the tibial nerve with a cutoff of 71.3 kPa was the most sensitive (68.3%) and had a higher area under the curve (0.712; 0.602-0.806) among the 3 shear elasticity indices for diagnosing DPN when used alone. When combining SWE with TCSS in diagnosing DPN, the most effective parameter was the EMax, which yielded a sensitivity of 100.00% and a specificity of 95.24%.SWE is a better diagnostic tool for DPN than the conventional ultrasonic parameter CSA, and a higher diagnostic value is attained when combining SWE with TCSS.

Clinical Significance of microRNA-146a in Patients with Ulcerative Colitis.

OBJECTIVE: To investigate the clinical significance of microRNA-146 (miRNA-146) in patients with ulcerative colitis (UC). METHODS: The present prospective observational study included 312 UC patients from August 2015 to August 2018. All patients were divided into mild/moderate and severe groups according to their Sutherland Disease Activity Index (DAI) scores. The clinical activity index and endoscopic index were used to determine the severity of UC. Serum levels of NF-κB, CRP, IL-1ß, IL-6, and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). The expression of miRNA-146a was measured using RT-qPCR. RESULTS: The expression of miRNA-146a was significantly lower in both mild/moderate and severe UC patients than the healthy control, and the severe UC patients had the lowest level of miRNA-146a. All inflammatory factors were remarkably higher in severe UC patients. miRNA-146a level was negatively correlated with NF-κB, CRP, IL-1ß, IL-6 and TNF-α levels. The ratio of severe UC patients was significantly higher in patients with lower miRNA-146a than in patients with higher miRNA-146a. The levels of NF-κB, CRP, IL-1ß, IL-6, and TNF-α were all remarkably higher in patients with lower miRNA-146a. Patients with lower miRNA-146a had higher Sutherland DAI scores, clinical activity index, and endoscopic index. CONCLUSIONS: miRNA-146a was down-regulated in UC patients and was negatively correlated with serum levels of inflammatory factors as well as severity of UC patients.

TLR activation inhibits the osteogenic potential of human periodontal ligament stem cells through Akt signaling in a Myd88- or TRIF-dependent manner.

BACKGROUND: This study investigated the effects of Toll-like receptors (TLRs) on human periodontal ligament stem cells (hPDLSCs) osteogenic differentiation and the associated mechanisms. METHODS: TLR1, TLR3, TLR4, and TLR6 expression in hPDLSCs was evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and flow cytometry, whereas their functional roles were assessed based on nuclear factor (NF)-κB activation and proinflammatory cytokine expression. The osteogenic effects of these TLRs were analyzed by alkaline phosphatase (ALP) staining, ALP activity, and alizarin red staining. The roles of Myd88, TRIF, and downstream molecules mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt) in TLR-mediated impaired osteogenic differentiation were examined by real-time RT-PCR and western blotting using specific small interfering RNA siRNA and pharmacologic inhibitors. The involvement of Akt activation in restoring TLR1-, 4-, and 6-mediated osteogenic suppression was verified using the Akt activator SC-79. RESULTS: TLR1, TLR3, TLR4, and TLR6 were highly expressed functionally in hPDLSCs and high doses of TLR ligands inhibited osteogenic potential. Furthermore, blocking Myd88 partly rescued the decrease in osteogenesis mediated by TLR1, TLR4, and TLR6 activation by enhancing Akt phosphorylation; likewise, TRIF suppression partially rescued lipopolysaccharide (LPS)-mediated osteogenic inhibition through ERK and Akt activation. Moreover, Akt activation restored the TLR-mediated inhibition of hPDLSC osteogenic differentiation. CONCLUSIONS: High doses of TLR1, TLR4, and TLR6 ligands suppress hPDLSC osteogenic differentiation by inhibiting Akt activation through Myd88- or TRIF-dependent signaling pathways. Blocking these adaptors or reactivating Akt could restore the TLR-mediated decrease in hPDLSC osteogenesis, and might be an ideal strategy for periodontitis treatment.

A novel mutation of MSX1 in oligodontia inhibits odontogenesis of dental pulp stem cells via the ERK pathway.

BACKGROUND: Tooth agenesis, one of the most common developmental anomalies, can affect the function and esthetics of patients. The aim of the present study was to identify genetic clues for familial tooth agenesis and explore the underlying mechanisms, focusing on the role of human dental pulp stem cells (hDPSCs). METHODS: We applied Sanger sequencing to identify the cause of oligodontia in a Chinese family. DNA transfection and functional analysis in DPSCs was also performed to explore the impact of the identified mutation on this phenotype. RESULTS: In this study, a novel frameshift mutation, the twenty-nucleotide deletion (c.128_147del20, p.Met43Serfsx125), in exon1 of MSX1 was detected in a Chinese family causing autosomal dominant nonsyndromic oligodontia. The mutation cosegregated with the tooth agenesis phenotype in this family. DPSCs transfected with mutant MSX1 plasmid showed decreased capacity of osteo/odontogenic differentiation with a lower expression level of dentin sialophosphoprotein (DSPP) and bone sialoprotein (BSP) compared with those transfected with control MSX1 plasmid. Mechanically, control MSX1 showed nuclear localization while the mutant MSX1 inhibited its nuclear translocation and localized on the cytoplasm to inhibit ERK phosphorylation. Furthermore, we inhibited the ERK pathway using ERK inhibitor (U0126) treatment in control MSX1-transfected DPSCs which could downregulate mineralized nodule formation and the expression of odontogenic genes. CONCLUSION: We demonstrated a novel MSX1 mutation causing familial nonsyndromic oligodontia and mechanically MSX1 regulates odontogenesis through the ERK signaling pathway in human dental pulp stem cells.

Transcriptional activation of glucose transporter 1 in orthodontic tooth movement-associated mechanical response.

The interplay between mechanoresponses and a broad range of fundamental biological processes, such as cell cycle progression, growth and differentiation, has been extensively investigated. However, metabolic regulation in mechanobiology remains largely unexplored. Here, we identified glucose transporter 1 (GLUT1)-the primary glucose transporter in various cells-as a novel mechanosensitive gene in orthodontic tooth movement (OTM). Using an in vivo rat OTM model, we demonstrated the specific induction of Glut1 proteins on the compressive side of a physically strained periodontal ligament. This transcriptional activation could be recapitulated in in vitro cultured human periodontal ligament cells (PDLCs), showing a time- and dose-dependent mechanoresponse. Importantly, application of GLUT1 specific inhibitor WZB117 greatly suppressed the efficiency of orthodontic tooth movement in a mouse OTM model, and this reduction was associated with a decline in osteoclastic activities. A mechanistic study suggested that GLUT1 inhibition affected the receptor activator for nuclear factor-κ B Ligand (RANKL)/osteoprotegerin (OPG) system by impairing compressive force-mediated RANKL upregulation. Consistently, pretreatment of PDLCs with WZB117 severely impeded the osteoclastic differentiation of co-cultured RAW264.7 cells. Further biochemical analysis indicated mutual regulation between GLUT1 and the MEK/ERK cascade to relay potential communication between glucose uptake and mechanical stress response. Together, these cross-species experiments revealed the transcriptional activation of GLUT1 as a novel and conserved linkage between metabolism and bone remodelling.

Declined Expression of Histone Deacetylase 6 Contributes to Periodontal Ligament Stem Cell Aging.

BACKGROUND: Identification of regulators for aging-associated stem cell (SC) dysfunctions is a critical topic in SC biology and SC-based therapies. Periodontal ligament stem cell (PDLSC), a kind of dental mesenchymal SC with dental regeneration potential, ages with functional deterioration in both in vivo and ex vivo expansion. However, little is known about regulators for PDLSC aging. METHODS: Expression changes of a potential regulator for PDLSC aging, histone deacetylase 6 (HDAC6), were evaluated within various models. Senescence-associated phenotypic and functional alternations of PDLSC in loss-of-function models for HDAC6 were examined using HDAC6-specific pharmacologic inhibitors or RNA interference-based knockdown. Involvement of p27Kip1 in HDAC6-associated aging was demonstrated by its acetylation and stability changes along with overexpression or functional inhibition of HDAC6. RESULTS: Expression of HDAC6 decreased significantly in replicative senescence and induced SC aging models. Loss-of-function experiments suggested that pharmacologic inhibition of deacetylase activity of HDAC6 accelerated PDLSC senescence and impaired its SC activities, which showed reduced osteogenic differentiation and diminished migration capacities. Examination of markers for proliferative exhaustion of SCs revealed that protein level of p27Kip1 was specifically elevated after HDAC6 inhibition. HDAC6 physically interacted with p27Kip1 and could deacetylate p27Kip1. Importantly, acetylation of p27Kip1 was negatively regulated by HDAC6, which correlated with alteration of p27Kip1 protein levels. CONCLUSION: Data suggest that HDAC6 plays an important role in PDLSC aging, which is dependent, at least partially, on regulation of p27Kip1 acetylation.

Transcriptional regulation of A33 antigen expression by gut-enriched Krüppel-like factor.

A33 antigen is a membrane-bound protein that is expressed only in intestinal epithelium and in most human colon cancers. Thus, A33 antigen has been explored as a potential therapeutic target for the treatment of colon cancers. However, little is known about the mechanism responsible for the tissue-specific pattern of its expression. In this report, we demonstrate that gut-enriched Krüppel-like factor (GKLF) binds to the promoter region of A33 antigen gene in colonic carcinoma cells and that mutations in the GKLF binding sequence in this region lead to diminished expression of A33 antigen. In addition, the expression of GKLF is linked to the expression of A33 antigen and blocking the expression of GKLF leads to the abolishment of A33 antigen expression. These results suggest that GKLF is a critical regulator in inducing the expression of A33 antigen in intestinal epithelium. While it has been suggested that GKLF is a regulator in inducing cell growth arrest and differentiation of the intestine, our observation that A33 antigen gene is a downstream target for GKLF suggests a more complex and diverse role for GKLF in the gut.

The largely normal response to Toll-like receptor 7 and 9 stimulation and the enhanced expression of SIGIRR by B cells in systemic lupus erythematosus.

BACKGROUND: Altered Toll-like receptor (TLR) signaling has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The present study was undertaken to characterize responses of B cells from SLE patients to TLR7 and TLR9 stimulation and to explore the potential role of single immunoglobulin interleukin-1 receptor related molecule (SIGIRR) in the regulation of TLR-mediated responses of SLE B cells. METHODOLOGY/PRINCIPAL FINDINGS: Peripheral blood mononuclear cells (PBMC) were isolated from 64 patients with SLE and 37 healthy donors. CD19+ B cells purified using microbeads were cultured with TLR7 or TLR9 agonists. Cell proliferation was measured by thymine incorporation and the frequency of antibody-secreting cells was determined by ELISPOT assay. SIGIRR expression in PBMCs and B cells was analyzed using flow cytometry analysis. In contrast to the enhanced proliferation following B cell receptor (BCR) engagement, B cells from SLE patients exhibited a virtually normal proliferative response to TLR7 or TLR9 stimulation. Moreover, B cells from SLE patients and healthy donors were almost equally competent to differentiate into antibody-secreting cells upon TLR engagement except for a reduction in the generation of IgG-secreting cells by TLR9-stimulated lupus B cells. In line with these somehow unexpected observations, SLE B cells were found to express a significantly higher level of SIGIRR than normal B cells. CONCLUSIONS/SIGNIFICANCE: Despite the reported upregulation of TLR7 and TLR9 expression in B cell from SLE patients, their responses to TLR stimulation were largely normal. The increased expression of the negative regulator SIGIRR may be partly responsible for the "balance of terror".