Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) influences adipocytes injuries through triggering XBP1 and activating mitochondria-mediated apoptosis.

Obesity is an important public-health problem worldwide. This study aimed to determine effects of porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on adipocytes injuries and explore associated mechanisms. Adipocytes were isolated from SD rats. pLVX-XBP1 (XBP1 over-expression) and pLVX-XBP1-RNAi (silencing XBP1) were structured and transfected into adipocytes. All adipocytes were divided into pLVX-NC, pLVX-XBP1, pLVX-NC+Pg-LPS and pLVX-XBP1+ Pg-LPS group. Oil-Red O staining was employed to identify isolated adipocytes. Quantitative real-time PCR (qRT-PCR) was used to examine gene transcription of IL-6, TNF-α, leptin, adiponectin. Western blotting was used to detect Bax and caspase-3 expression. Adipocytes were successfully isolated and identified with Oil-Red O staining. Both XBP1 mimic and XBP1 RNAi were effectively transfected into adipocytes with higher expressing efficacy. XBP1 over-expression significantly aggravated Pg-LPS induced inflammatory response compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS significantly enhanced leptin and inhibited adiponectin expression by up-regulating XBP1 expression (p<0.05). XBP1 silence significantly alleviated Pg-LPS induced inflammatory response and reduced leptin, enhanced adiponectin expression in Pg-LPS treated adipocytes compared to adipocytes without Pg-LPS treatment (p<0.05). Pg-LPS induced apoptosis of adipocytes by enhancing XBP1 expression and modulating Bcl-2/Bax pathway associated molecules. In conclusion, Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) induces adipocytes injuries through modulating XBP1 expression and initialling mitochondria-mediated apoptosis.

[Analysis of clinical aesthetic effect of buccal alveolar ridge preservation and connective tissue transplantation with single implant].

PURPOSE: To evaluate the clinical aesthetic effect of buccal alveolar ridge preservation (ARP) and connective tissue transplantation (CTG) in patients who received a single implant. METHODS: Forty-three patients with tooth loss admitted to the Department of Stomatology of Shunde Hospital of Southern Medical University from May 2014 to May 2016 were included in the study. Tooth extraction, ARP, implant implantation, CTG and permanent repair were performed respectively. The incidence of bleeding, depth of probing, marginal bone resorption, and red-white aesthetic effect of implants were evaluated 1 year and 3 years after surgery. The buccal mucosa thickness of implants before, immediately after CTG, 1 year and 3 years after surgery were measured. The patient satisfaction was evaluated by visual analogue scale (VAS) from masticatory function, overall aesthetics, attachment height, and color, respectively. The implant conditions at the third year after surgery were observed, and complications during follow-up were recorded. SPSS 20.0 software package was used for statistical analysis of the data. RESULTS: The follow-up rate in the first year after surgery was 100%, and that in the third year after surgery was 90.70%. One year and 3 years after operation, the aesthetic effect of the implant was satisfactory. At the 3rd year after operation, the scores of the near middle gingival papillary were significantly higher than that at the 1st year after operation (P<0.05). The buccal mucosal thickness of the implant immediately after CTG and 1 year and 3 years after surgery increased significantly compared with that before CTG (P<0.05). The buccal mucosal thickness of the implant increased 1.02 mm (relative stability: 90.12%) 1 year after operation and 1.01 mm (relative stability: 84.31%) 3 years after operation, respectively. The satisfaction scores of the patients on chewing function, overall aesthetics, attachment height and color of the implant immediately after CTG, one year after surgery and 3 years after surgery were all > 8. The 3-year survival rate of the implants was 100%, and the 3-year success rate of the implants was 97.44%. During the follow-up, two patients developed peri-implant mucositis, which was relieved after tooth cleaning, but no complications such as tissue flap necrosis, limited opening and tongue movement disorder occurred. CONCLUSIONS: ARP and CTG have good clinical and aesthetic effects on patients with tooth loss. In three years, the buccal mucosal thickness of the implant can be increased and relatively stable, which is worthy of clinical promotion and application.

Coxsackievirus A10 atomic structure facilitating the discovery of a broad-spectrum inhibitor against human enteroviruses.

Coxsackievirus A10 (CV-A10) belongs to the Enterovirus species A and is a causative agent of hand, foot, and mouth disease. Here we present cryo-EM structures of CV-A10 mature virion and native empty particle (NEP) at 2.84 and 3.12 Å, respectively. Our CV-A10 mature virion structure reveals a density corresponding to a lipidic pocket factor of 18 carbon atoms in the hydrophobic pocket formed within viral protein 1. By structure-guided high-throughput drug screening and subsequent verification in cell-based infection-inhibition assays, we identified four compounds that inhibited CV-A10 infection in vitro. These compounds represent a new class of anti-enteroviral drug leads. Notably, one of the compounds, ICA135, also exerted broad-spectrum inhibitory effects on a number of representative viruses from all four species (A-D) of human enteroviruses. Our findings should facilitate the development of broadly effective drugs and vaccines for enterovirus infections.

Associations among glycemic excursions, glycated hemoglobin and high-sensitivity C-reactive protein in patients with poorly controlled type 2 diabetes mellitus.

The aim of the present study was to explore the associations among glycemic excursions, glycated hemoglobin (HbA1c) and high-sensitivity C-reactive protein (hs-CRP) in patients with poorly controlled type 2 diabetes mellitus (T2DM) using a continuous glucose monitoring system (CGMS). Sixty-three patients with T2DM whose HbA1c levels were >7% wore a CGMS device for 72 h. According to their HbA1c levels, patients were divided into three groups as follows: Group A (HbA1c ≤9.32%), group B (9.32%< HbA1c ≤11.76%) and group C (HbA1c >11.76%). Patients were also divided into two groups according to the mean amplitude of glycemic excursions (MAGE) as follows: Low glycemic excursion group (MAGE, <3.9 mmol/l) and high glycemic excursion group (MAGE, ≥3.9 mmol/l). Clinical data and the hs-CRP levels in different groups were compared. No significant difference was observed in the MAGE among groups A, B and C (P>0.05). The level of hs-CRP was significantly higher in group C compared with that in groups A and B, and in group B compared with that in group A (P<0.05). Multivariate stepwise regression analysis indicated that HbA1c correlated with hs-CRP (P<0.05). MAGE and HbA1c were independent indices for the assessment of glycemic control. In addition, HbA1c had a considerable effect on the serum hs-CRP level.

Primary prevention of macroangiopathy in patients with short-duration type 2 diabetes by intensified multifactorial intervention: seven-year follow-up of diabetes complications in Chinese.

OBJECTIVE: To explore whether intensified, multifactorial intervention could prevent macrovascular disease in patients with recently diagnosed type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 150 type 2 diabetic patients, with disease duration of <1 year and without clinical arteriosclerotic disease or subclinical atherosclerotic signs confirmed by ultrasonographic scanning of three conducting arteries, were randomized into an intensive intervention group and a conventional intervention group. They then received intensive, multifactorial intervention or conventional intervention over 7 years of follow-up. The patients' common carotid intima-media thicknesses (CC-IMTs) were measured every year. The primary outcome was the time to the first occurrence of CC-IMTs ≥1.0 mm and/or development of atherosclerosis plaques in the carotid artery. The secondary outcome was clinical evidence of cardiovascular disease. RESULTS: A total of 70 patients in the intensive group and 68 patients in the conventional group completed the 7-year follow-up. Subclinical macrovascular (primary) outcomes occurred in seven cases in the intensive group and 22 cases in the conventional group for a cumulative prevalence of 10.00 and 32.35%, respectively (P < 0.05). No significant differences between the two groups were observed regarding the secondary outcome. CONCLUSIONS: Primary prevention of macrovascular diseases can be achieved through intensified, multifactorial intervention in patients with short-duration type 2 diabetes. Type 2 diabetic patients should undergo intensive multifactorial interventions with individual targets for the prevention of macrovascular diseases.

[Effects of Simvastatin on osteoblast activity of human periodontal ligament cells].

OBJECTIVE: To study the effects of Simvastatin on the osteoblast activity of human periodontal ligament (PDL) cells. METHODS: The third passage human PDL were cultured in conditional mineralization medium with different concentrations of Simvastatin. The cells were divided into A group (0 mol/L), B group (1 x 10(-9) mol/L), C group (1 x 10(-8) mol/L), D group (1 x 10(-7) mol/L) and E group (1 x 10(-6) mol/L). Alkaline phosphatase (ALP) activity, osteopontin (OPN) and capability of mineralization were measured. RESULTS: Differentiation osteoblast and mineralization of human PDL were improved in all treatment groups with different concentrations of Simvastatin (1 x 10(-9), 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L). Compared with control group, statistically significant differences were found in 1 x 10(-8) mol/L, 1 x 10(-7) mol/L and 1 x 10(-6) mol/L groups (P<0.05). The maximum effect was observed at the concentration of 1 x 10(-7) mol/L. CONCLUSION: Optimal concentration of Simvastatin can improve the osteoblastic activity of human PDL.

[Expression and purification of human amelogenin mature peptide in Escherichia coli].

OBJECTIVE: To establish the expression and purification route for human amelogenin mature peptide in Escherichia coli and obtain the purified amelogenin (AMG) mature peptide. METHODS: Recombined plasmid pGEX-4T-1-AMG was transformed to Escherichia coli BL21. After expression, AMG was purified with glutathione S-transferase fusion protein purification system (GSTrapFF) column. RESULTS: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization results showed that 45,000 GST-AMG fusing protein and 19,000 target AMG mature peptide were obtained successfully. CONCLUSIONS: pGEX-4T-1-AMG-BL21 system is used successfully to express and purify human AMG mature peptide.

[Effects of simvastatin on the immune-related genes of mouse osteoblasts].

OBJECTIVE: To investigate the expression of immune-related genes during osteogenesis stimulated by simvastatin. METHODS: After treated with simvastatin, the expression of immune-related genes of mouse osteoblast was examined with gene chip (BiostarM-140s). RESULTS: There were 16 differently expressed genes related to immune function, with nine down-regulated genes and seven up-regulated genes. CONCLUSIONS: After treated with simvastatin, expression of inflammation related genes is down-regulated and inflammation inhibitor genes is up-regulated in mouse osteoblasts.

[Distribution of phosphorus species in the sediment from Weishanhu district in Lake Nansihu].

In order to learn the phosphorus (P) species distribution in sediments of Lake Nansihu, 0-25 cm sediments from Weishanhu district in Lake Nansihu were analyzed with a sequential extraction method. The results show that: the average values of Ex-P, Al-P, Fe-P, Oc-P, Ca-P, De-P, Org-P in Weishanhu district sediments are 5.62 mg/kg, 4.08 mg/kg, 12.25 mg/kg, 13.34 mg/kg, 116.67 mg/kg, 232.36 mg/kg and 396.79 mg/kg respectively, and the rank order of P-fractionation for Lake Nansihu is Al-P < Ex-P < Fe-P < Oc-P < Ca-P < De-P < Org-P. The vertical phosphorous species distribution exhibits the obvious rule that exchangeable P (Ex-P), Fe-bound P (Fe-P), occluded P (Oc-P), organic P (Org-P) value decrease with depth, while the values of Al-bound P (Al-P), authigenic calcium bound P (A Ca-P), detrital apatite P (De-P) increase. The Sum1 content (the sum of Ex-P, Al-P and Fe-P) in surficial sediments is remarkably positively correlated with the PO4(3-) concentration of overlaying water, to which Fe-P content contributes the most with the correlation index 0.72. In spatial phosphorous species distribution, the discrepancy of potential active species(Ex-P, Al-P and Fe-P) is greater than those inert ones (Oc-P, Ca-P and De-P), and Org-P is only second to those potential active species.

[Flocculation characteristics of the return sludge in chemical-biological flocculation process].

The chemical-biological flocculation (CBF) process was used to treat municipal wastewater in Shanghai, and the effect of its return sludge on pollutant removal was studied through stopping chemical addition, simulating different return sludge ratio, and analyzing extracellular polymeric substances (EPS). The results show that the return sludge in CBF exhibits strong pollutant removal capability, and chemical in its return sludge can further TP removal and enhance CBF's adaptability to the influent TP impulse. After stopping PAFC (Al2 O3 : 10.8%, Fe2 O3: 1.8%) addition, COD removal efficiency is achieved at about 50%, and removal rates of PO4(3-) and TP gradually decline. It is also found that chemical addition plays an important role in promote sludge sedimentation in CBF. EPS extracted from CBF is 145.89 mg/g, while EPS from CEPT is only 17.24 mg/g, which indicates that there is strong biological flocculation behavior in CBF. With the increase of return sludge ratio, TP and COD removal efficiencies decrease in CEPT, which reveals that chemical in waste sludge has poor flocculation capability, while CBF return sludge exhibits good flocculation behavior due to its biological flocculation. Chemical flocculation and biological flocculation work collaboratively in CBF process.

[Primary preventive effect of metformin upon atherosclerosis in patients with type 2 diabetes mellitus].

OBJECTIVE: To investigate the preventive action of metformin for atherosclerosis (AS) in patients with type 2 diabetes mellitus (T2DM). METHODS: A total of 140 cases with T2DM were assigned to 2 groups taking metformin (n = 75) or not (n = 65). All cases received intensive control of blood glucose, blood pressure and blood lipids for 100 weeks. The data before and after intensive control were recorded and statistically analyzed. Common carotid intima-media thickness (CC-IMT) was the efficacy endpoint of AS. RESULTS: Diastolic blood pressure (DBP), fasting blood glucose, post 2-hour blood glucose, glycated hemoglobin A1c, triglyceride (TG) and total cholesterol (TC) decreased in both groups after intensive metabolic control for 100 weeks (P < 0.05). Body mass index (BMI), HOMA insulin resistance index (HOMA-IR) and CC-IMT decreased in metformin group (P < 0.05) while high-density lipoprotein cholesterol (HDL-C) increased (P < 0.05). BMI (23.1 +/- 0.98) kg/m2, HOMA-IR (1.68 +/- 0.20) and CC-IMT (0.55 +/- 0.13) mm in metformin group were lower than those in non-metformin group [(24.1 +/- 0.05) kg/m2, 2.03 +/- 1.38, (0.70 +/- 0.15) mm)] at 100 week (P < 0.05). CC-IMT was positively correlated with BMI (r = 0.489, P < 0.05) , TC (r = 0.429, P < 0.05), low-density lipoprotein cholesterol (LDL-C) (r = 0.426, P < 0.05) and HOMA-IR (r = 0.428, P < 0.05). CONCLUSION: Metformin attenuates CC-IMT of patients with T2DM and it has the primary preventive effect upon AS in patients with T2DM.

[Clinical application of maxillary sinus lift with Summers osteotome].

OBJECTIVE: To introduce and evaluate the procedure and the effect of maxillary sinus lift with closed technique by Summers osteotome, bone grafting and simultaneous implant placement. METHODS: 66 cases with severely resorbed alveolar bone in maxillary posterior region received sinus lift with Summers osteotome, simultaneously bone grafting and implants placement. The final restoration was finished at 6 months postoperatively. RESULTS: The operation procedure were eventless in the 66 cases, the sinus floor were elevated by 2-5 mm, three-dimensional reconstruction of the CT scan pictures showed the smooth dome profile of the lifting sites and no signs of laceration on the membrane, and there were no maxillary antritis after operation. After 6 months, no significantly bone graft resorption and good osseointegration were noticed in X-ray imaging. The final restoration was finished at this time. 12-24 months after the restoration, all implants inserted were remain, the hard and soft tissue were healthy, prosthesis were stable and functioned. X-ray showed good osseointegration in the lifting sites, the vertical resorption around the implants were less than 1 mm. CONCLUSION: With properly use of Summers osteotome, scraps of the bone in the implant sockets can be pushed into the sinus, these autogenous bone scraps were in favor of the osseogenesis and the sinus floor can be easily elevated by the method with very infrequent complications. It enlarged indication of dental implants and avoided operation of harvesting autogenous bone in other site. The method is simple and valuable to clinical application.

[Expression and purification of human amelogenin in Escherichia coli].

OBJECTIVE: To establish the expression and purification route for the gene encoding human amelongenin (AMG) mature peptide in Escherichia coli (E. coli). METHODS: Recombined plasmid pGEX-4T-1/AMG was identified by double endonuclease digestion electrophoretogram and DNA sequence analysis. The recombined plasmid was transformed to E. coli BL21. The inducing time, isopropyl-beta-D-thiogalactopyranoside (IPTG) concentration and inducing temperature were optimized for the express system. Under the optimized condition, the target fusing protein in superatant, periplasm, plasm and inclusion body was analyzed separately. A great amount of target fusing protein was found in the dissoluble protein. AMG fusing protein was purified by the GSTrapFF affinity column. RESULTS: Double endonuclease digestion electrophoretogram and DNA sequence analysis were done to identify the recombined vector pGEX-4T-1/AMG. The results were consistent with the anticipation. The optimum inducing time was 14.5 hours. The optimum IPTG concentration was 1.0 mmol/L. The optimum inducing temperature was 20 degrees C. Under this condition, the target protein was expressed to a maximum. Plentiful target protein was expressed in plasm and inclusion body under the optimized condition. A mount of plasm protein was obtained and purified by the GSTrapFF affinity column. The purified liquid was collected and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PACE). The protein electrophoresis map showed that AMG fusing protein was purified successfully. After twice elution, high pure fusing protein was obtained. CONCLUSION: pGEX-4T-1/AMG system is used successfully to express human AMG fusing protein.

[Construction of the prokaryotic expression vector for human amelogenin gene].

PURPOSE: To construct the prokaryotic expression vector for the gene encoding human amelogenin mature peptide. METHODS: The DNA sequence encoding human amelogenin was inserted into the plasmid pMD 18-T by TA clone technique. pMD 18-T/AMG was digested by BamH I and Xho I. Target DNA was cloned into Prokaryotic expression plasmid pGEX-4T-1 by T4 DNA ligase RESULTS: Double endonuclease digestion electrophoresis and DNA sequence analysis was conducted to identify the recombined vector pMD 18-T/AMG and pGEX-4T-1/AMG. The results were consistent with the anticipation. CONCLUSION: The prokaryotic expression vector for the gene encoding human amelogenin mature peptide is constructed successfully.

A single tetracycline-regulated vector devised for controlled insulin gene expression.

OBJECTIVE: To construct a single plasmid vector mediating doxycycline-inducible recombined human insulin gene expression in myotube cell line. METHODS: An expression cassette of rtTAnls driven by promoter of human cytomegalovirus and a furin-cuttable recombined human insulin expression cassette driven by a reverse poly-tetO DNA motif were cloned into a single plasmid vector (prTR-tetO-mINS). The prTR-tetO-mINS and pLNCX were co-transfected into a myotube cell line (C2C12) and pLNCX vector were used as a control. After selection with G418, the transfected cells were induced with doxycycline at concentrations of 0, 2, and 10 microg/mL. RT-PCR was used to determine expression levels of recombinant insulin mRNA at the 5th day. Insulin production in cell cultures medium (at different incubation time) and cell extracts (at the 7th day) were analyzed with human pro/insulin RIA kits. RESULTS: Immune reactive insulin (IRI) level in cell medium was found increased at 24 hours of doxycycline incubation, and still increased at the 5th day. After withdrawn of doxycycline, IRI decreased sharply and was at baseline three days later. IRI and human insulin mRNA levels were positively related to different levels of doxycycline. A 25-fold increase in IRI was found against background expression at the 7th day. CONCLUSION: Human insulin expression can be successfully regulated by doxycycline and the background was very low. This single tet-on insulin expression system may provide a new approach to a controlled insulin gene therapy in skeletal muscle.