Synthesis and receptor dependent 4D-QSAR studies of 4,5-dihydro-1,3,4-oxadiazole derivatives targeting cannabinoid receptor.

Cannabinoid receptor has been shown to be overexpressed in various types of cancers, especially non-small cell lung cancer. As a result, it could be used as novel target for anticancer treatments. Because receptor-dependent 4D-QSAR generates conformational ensemble profiles of compounds by molecular dynamics simulations at the binding site of the enzyme, this work describes the synthesis, biological activity evaluation and 4D-QSAR studies of 4,5-dihydro-1,3,4-oxadiazole derivatives targeting cannabinoid receptor. Compared with WIN55,212-2, compound 5 f showed the best antiproliferative activity. The receptor-dependent 4D-QSAR model was generated by multiple linear regression method using QSARINS. Leave-n-out cross-validation and chemical applicability domain were performed to analyse the independent test set and to verify the robustness of the model. The best 4D-QSAR model showed the following statistics: r2 = 0.8487, Q2LOO = 0.7667, Q2LNO = 0.7524, and r2Pred = 0.8358.

[The definition of heat-wave based on mortality risk assessment in different regions of China].

Objective: To identify the definition of heat wave based on mortality risk assessment in different regions of China. Methods: Daily mortality (from China Information System for Disease Control and Prevention) and meteorological data (from National Meteorological Information Center in China) from 66 counties with a population of over 200 000 were collected from 2006-2011. With the consideration of climate type and administrative division, China was classified as seven regions. Firstly, distributed lag non-linear model (DLNM) was used to estimate community-specific effects of temperature on non-accidental mortality. Secondly, a multivariate meta-analysis was applied to pool the estimates of community-specific effects to explore the region-specific temperature threshold and the duration for definition of heat wave. Results: We defined regional heat wave of Northeast, North, Northwest, East, Central and Southwest China as being two or more consecutive days with daily mean temperature higher than or equal to the P(64), P(71), P(85), P(67), P(75) and P(77) of warm season (May to October) temperature, respectively, while the thresholds of temperature were 21.6, 23.7, 24.3, 25.7, 28.0 and 25.3 ℃. The heat wave in South China was defined as five or more consecutive days with daily mean temperature higher than or equal to the P(93) (30.4 ℃) of warm season (May to October) temperature. Conclusion: The region-specific definition of heat wave developed in our study may provide local government with the guidance of establishment and implementation of early heat-health response systems to address the negative health outcomes due to heat wave.

[Commuting mode specific exposure to PM(2.5) in urban area of Guangzhou].

Objective: To find the differences in PM(2.5) exposure level in the context of four commuting modes (by walk, bicycle, bus and subway) in Guangzhou. Methods: The PM(2.5) exposure assessment was carried out from January to December 2015 in Guangzhou. PM(2.5) was measured by using SidePak individual dust meter (AM510, TSI Inc. USA) with time interval of 1 minute. Our measurement was taken on Monday, Wednesday, Friday and Sunday in the second week of each month and the samples were collected in the morning (07:00-09:00), afternoon (11:00-13:00) and evening (17:00-19:00). Results: A total of 284 air samples during walking, 281 air samples during bicycle riding, 278 air samples in bus, and 280 air samples in subway were collected. The median PM(2.5) concentrations exposed during walking, during bicycle riding, in bus and in subway were 38.4, 38.6, 23.3 and 24.1 µg/m(3), respectively, which were positive correlated with exposure concentration in fixed surveillance sites. The exposure level was lowest in summer, and highest in winter. The median of one-way exposure level to PM(2.5) from high to low were as follows: 21.0 µg for bicycle riding, 20.1 µg for walking, 5.1 µg for taking bus and 2.6 µg for taking subway. The season and time specific one-way exposure levels to PM(2.5) of four commuting modes were consistent. Conclusions: The exposure level to PM(2.5) was obviously higher during walking and bicycle riding than that in bus and subway. The exposure level to PM(2.5) during walking was higher than that during bicycle riding, in bus and in subway.

[Association of job burnout with subjective well-being and health status among employees from 29 provinces in China].

Objective: To investigate the influence of job burnout on subjective well-being and health status among employees in China. Methods: The data from the 2014 China Labor-force Dynamic Survey were used to analyze the association of job burnout with subjective well-being and health status among 7289 employees aged 18-64 years from 29 provinces in China.Some items from the Maslach Burnout Inventory-General Survey were used to investigate job burnout; subjective well-being assessment included life happiness and degree of satisfaction with living condition; the questions for self-evaluation of health status were used to analyze health status. Results: Of all employees,30.5% had low subjective well-being and 4.7% had poor health status based on self-evaluation. The logistic regression analysis showed that emotional exhaustion(two items), reduced sense of personal accomplishment,and cynicism were risk factors for low subjective well-being(OR=1.07,1.11,1.10,and 1.06,P<0.001),and emotional exhaustion(two items)was a risk factor for poor health status (OR=1.10 and 1.07,P<0.001).Reduced sense of personal accomplishment and cynicism had no significant influence on health status(P>0.05). Conclusion: Emotional exhaustion is a major influencing factor for health status,and reducing job burnout may be an effective method for improving subjective well-being and health status.

[Effectiveness of health education about heat wave hazard prevention in the elderly: a mixed effect model analysis].

Objective: To evaluate the effectiveness of health education about prevention of heat wave hazard in the elderly. Methods: A non-randomized controlled trial was conducted during the summer of 2015 among a sample of residents aged ≥60 years in Panyu district, Guangzhou. Eight intervention measures for heat wave hazard prevention were taken in intervention group for 3 months (from August to October) and in control group no intervention measures were taken. The comparison of intervention effects was conducted between the intervention group and control group with mixed effect model after the collection of related information with same questionnaire. Results: After adjusting of family per capita income, family air-condition availability, alcohol use, disease history and time, the average score of risk awareness in the intervention group increased by 1.62, while it was 0.51 in the control group, the difference was significant (t=2.76, P=0.006). A significant effect was observed in the intervention group on the reduction of hospitalizations due to chronic diseases. The hospitalization rate due to chronic diseases in resent 3 months in the intervention group decreased from 32.39% (46/142) before intervention to 28.87% (41/142) after intervention; while in the control group, it increased from 26.28% (41/156) before intervention to 36.53% (57/156) after intervention. There was no significant difference between the two groups in awareness of knowledge on heat wave hazard prevention and the score of adaptation to heat wave. Conclusion: Health education programs could improve the risk awareness on heat waves, and reduce the hospitalizations due to chronic diseases in the elderly.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells.

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Loss of macrophage migration inhibitory factor impairs the growth properties of human HeLa cervical cancer cells.

OBJECTIVES: This study aims to determine the role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with cell proliferation and tumour growth in vivo. MATERIALS AND METHODS: Our team used RNA interference technology to knock down MIF expression in human HeLa cervical cancer cells and to establish a stable cell line lacking MIF function. RESULTS: Our results showed that long-term loss of MIF had little effect on cell morphology, but significantly inhibited their population growth and proliferation. The HeLa MIF-knockdown cells retained normal apoptotic signalling pathways in response to TNF-alpha treatment; however, they exhibited unique DNA profiles following doxorubicin treatment, suggesting that MIF may regulate a cell cycle checkpoint upon DNA damage. Our data also showed that knockdown of MIF expression in HeLa cells led to increased cell adhesion and therefore impaired their migratory capacity. More importantly, cells lacking MIF failed to either proliferate in soft agar or form tumours in vivo, when administered to nude mice. CONCLUSION: MIF plays a pivotal role in proliferation and tumourigenesis of human HeLa cervical carcinoma cells, and may represent a promising therapeutic target for cancer intervention.

The support of matrix accumulation and the promotion of sheep articular cartilage defects repair in vivo by chitosan hydrogels.

OBJECTIVE: Chitosan has been widely used as an injectable scaffold in cartilage tissue engineering due to its characteristic biocompatibility and biodegradability. In this study, chitosan was used in its hydrogel form as a scaffold for chondrocytes that act to reconstruct tissue-engineered cartilage and repair articular cartilage defects in the sheep model. This study aims to find a novel way to apply chitosan in cartilage tissue engineering. METHODS: Temperature-responsive chitosan hydrogels were prepared by combining chitosan, beta-sodium glycerophosphate (GP) and hydroxyethyl cellulose (HEC). Tissue-engineered cartilage reconstructions were made in vitro by mixing sheep chondrocytes with a chitosan hydrogel. Cell survival and matrix accumulation were analyzed after 3 weeks in culture. To collect data for in vivo repair, reconstructions cultured for 1 day were transplanted to the freshly prepared defects of the articular cartilage of sheep. Then at both 12 and 24 weeks after transplantation, the grafts were extracted and analyzed histologically and immunohistochemically. RESULTS: The results showed that the chondrocytes in the reconstructed cartilage survived and retained their ability to secrete matrix when cultured in vitro. Transplanted in vivo, the reconstructions repaired cartilage defects completely within 24 weeks. The implantation of chitosan hydrogels without chondrocytes also helps to repair cartilage defects. CONCLUSIONS: The chitosan-based hydrogel could support matrix accumulation of chondrocytes and could repair sheep cartilage defects in 24 weeks. This study showcased the success of a new technique in its ability to repair articular cartilage defects.

Comparison of approaches for efficient gene silencing induced by microRNA-based short hairpin RNA and indicator gene expression.

MicroRNA-based short hairpin RNAs (shRNAs) are natural inducers of RNA interference and have been increasingly used in shRNA expression strategies. In the present study, we compared the efficiencies of exogenous green fluorescence protein (GFP) and endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) knockdown and red fluorescent protein (RFP) indicator expression mediated by three differently designed plasmids. RFP was introduced either at the 5' end, at the 3' end of the human mir155-based target gene (TG) (e.g., GFP or GAPDH) shRNA expression cassette (EC), or at the 3' end of the chimeric intron-containing TG shRNA EC. Comparisons with the control vector showed an obvious reduction of GFP or GAPDH expression with the various shRNA expression plasmids (P < 0.05). When RFP was located at the 5' end or at the 3' end of the TG shRNA EC, RFP expression was low; whereas when RFP was connected with the chimeric intron-containing TG shRNA EC, RFP expression was high. Taken together, this study demonstrated an efficient plasmid design for both TG silencing induced by microRNA-based shRNA and indicator gene expression in vitro.

A novel rat model of chronic heart failure following myocardial infarction.

In this report, we describe an improved method for the establishment of reproducible congestive heart failure (CHF) in a rat model. The area of myocardial infarction (MI) after ligation of the left anterior descending (LAD) coronary artery was quantified. Histological changes, heart function detected by echocardiography and isolated Langendorff perfusion, and selected biochemical factors were monitored after ligation of the LAD. Contrary to previous beliefs, thoracotomy in the second intercostal space provided a much better visualization of and easier access to the LAD and significantly reduced the mortality rate. Surface electrocardiogram (ECG) showed that the S-T interval was arched raised upward immediately after ligation. Typical morphological and functional changes of CHF were observed after LAD ligation. Cardiomyocytes in the infarcted zone were depleted and deranged. Biochemical analysis and enzyme-linked immunosorbent assay (ELISA) showed that superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and nitric oxide (NO) levels were significantly lowered in rats with MI than in the normal and sham groups, whereas serum malondialdehyde (MDA), MB isoenzyme of creatine kinase (CK-MB), cardiac troponin (cTnT) and C-reactive protein (CRP) levels were elevated. After MI, N-terminal pro-brain natriuretic peptide (NT-proBNP) was increased but insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF) in culture supernatant were lower than in the normal and sham groups. We present an improved model for maximal reproducibility of experimental CHF in rats which allows the study of molecular and physiological variables in relation to CHF.

A quick and efficient approach for gene silencing by using triple putative microRNA-based short hairpin RNAs.

The RNA interference (RNAi) technique has been widely used in gene function studies. It is typical to screen for effective siRNAs by knocking down targeted genes since a single gene can be suppressed by several siRNAs to varying degrees. The miRNA-based short hairpin RNA (shRNA) is a natural inducer of RNAi and has been used in siRNA expression strategies. We investigated the potential application of multiple putative microRNA-based shRNAs for gene silencing and studied the inhibition efficiency of exogenous GFP and firefly luciferase (luc) by triple human mir155-based shRNA expression vectors. A total of three candidate siRNA sequences targeted against GFP or luc were selected based on an online prediction program. Single and triple miRNA-155-based shRNAs targeted against GFP or luc were transfected into HEK293 cells mediated by the pcDNA(3) vector with an RNA polymerase II-type CMV (cytomegalovirus) promoter. Comparisons with negative control shRNAs revealed that GFP levels were markedly reduced by the triple miRNA-155-based GFP shRNA by fluorescent microscopy. Consistent results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based GFP shRNA significantly suppressed GFP expression (P < 0.01), without significant differences from the most effective single miRNA-155-based GFP shRNA (P > 0.05). Results from the dual luciferase assay and real-time quantitative RT-PCR revealed that the triple miRNA-155-based luc shRNA significantly suppressed luc expression as the most effective single miRNA-155-based luc shRNA (P < 0.05). These studies demonstrated the gene silencing efficiency mediated by the triple putative miRNA-155-based shRNAs. This suggested that multiple miRNA-based shRNAs are quick and valuable strategies for gene silencing.

Enhanced angiotensin converting enzyme 2 regulates the insulin/Akt signalling pathway by blockade of macrophage migration inhibitory factor expression.

BACKGROUND AND PURPOSE: Macrophage migration inhibitory factor (MIF) is now known to be a pro-inflammatory cytokine associated with insulin resistance. Our aim was to investigate whether angiotensin converting enzyme 2 (ACE2) could modulate the expression of MIF and the insulin/Akt-endothelial nitric oxide (NO) synthase (eNOS) signalling in a human endothelial cell line (EAhy926). EXPERIMENTAL APPROACH: A recombinant plasmid encompassing human ACE2 gene was constructed and transfected into the EAhy926 cells. The mRNA, phosphorylation and protein levels of p22phox, MIF, Akt and eNOS in endothelial cells were determined by real-time PCR and Western blot analysis, respectively. KEY RESULTS: Gene transfer of ACE2 suppressed the expression of p22phox and MIF induced by angiotensin (Ang) II and Ang IV, accompanied by a decreased level of malondialdehyde in cells. In addition, Ang II diminished insulin-stimulated phosphorylation of Akt (at Ser(473)) and eNOS (at Ser(1177)) and NO generation, effects which were reversed by ACE2 gene transfer and anti-MIF treatment in endothelial cells. CONCLUSIONS AND IMPLICATIONS: The results reveal that gene transfer of ACE2 regulated Ang II-mediated impairment of insulin signalling and involved the Akt-eNOS phosphorylation pathway. These beneficial effects of ACE2 overexpression appear to result mainly from blocking MIF expression in endothelial cells, suggesting that the ACE2 gene may be a novel therapeutic target for diseases related to inflammation and insulin resistance.

Fasting early morning rise in peripheral insulin: evidence of the dawn phenomenon in nondiabetes.

The dawn phenomenon, a tendency for glucose to rise between 0500 and 0800 h in subjects with diabetes, is also reflected as an increase in insulin required to maintain normoglycemia during closed-loop insulin infusion. Individuals without diabetes have minimal or absent rises in early morning glucose. To test the hypothesis that the absence of early morning glucose increases in subjects without diabetes is due to an increase in insulin levels, we measured insulin levels from 2400 to 0800 h in four male and two female volunteers. Subjects were on an unrestricted diet with three main meals and one bedtime snack at 2100 h. Blood samples were collected continuously in hourly pools by a constant-rate withdrawal pump. We observed the following: (1) hourly integrated concentration of glucose was stable from 2400 to 0800 h (range of mean plasma values, 94.5-97.3 mg/dl), and (2) hourly integrated concentration of insulin increased from the 0300-0400 (4.6 microU/ml) to the 0700-0800-h pool (6.2 microU/ml) (P less than 0.05). The observed increase in insulin in the early morning hours despite stable levels of glucose indicates a temporally increased insulin need in nondiabetic individuals similar to that found in individuals with diabetes. The mechanism underlying this increased insulin need may be similar in diabetes and nondiabetes, with the ensuing rise in glucose being dependent on the availability of compensatory insulin.