[Study on the relationship between non-alcoholic fatty liver disease and sarcopenia in type 2 diabetes mellitus].

Objective: To explore the relationship between non-alcoholic fatty liver disease and sarcopenia in patients with type 2 diabetes mellitus. Methods: 792 cases with type 2 diabetes mellitus who were hospitalized in the Endocrinology Department of the First Affiliated Hospital of Chongqing Medical University from June 2013 to December 2015 were enrolled in this cross-sectional study. Liver ultrasound examination and dual-energy X-ray absorptiometry (DXA) were used to examine the body composition. Patients were grouped according to gender and whether or not they had combined NAFLD, and indicators such as age, duration of diabetes, body mass index (BMI), waist circumference, biochemical indicators, skeletal muscle mass index (SMI), prevalence of sarcopenia, and medication status were collected. An independent-sample t-test, two-sample Kolmogorov-Smirnov test or χ (2) test were performed on the data. Logistic regression model was used to analyze the correlation between NAFLD, sarcopenia and SMI in diabetic patients of different genders. Results: The average age of 792 cases were (64.54 ± 9.61) years, and there were 301 (38%) patients with NAFLD. The prevalence of sarcopenia in male and female NAFLD patients was significantly higher than non-NAFLD patients (male 20.2% and 9.9%, χ(2) = 9.67, P = 0.002; female 12.2% and 5.1%, χ(2) = 5.64, P = 0.018). Male SMI (30.92 ± 2.31 and 31.81 ± 2.17, P < 0.001) and female SMI (25.48 ± 2.14 and 26.34 ± 2.28, P < 0.001) in NAFLD patients were significantly lower than non-NAFLD patients. Multivariate logistic regression analysis showed that sarcopenia was an independent risk factor for NAFLD in male patients with type 2 diabetes mellitus (OR = 2.006, 95% CI: 1.012 ~ 3.976, P = 0.046). There was no correlation between sarcopenia and NAFLD in female patients after adjusting for clinical risk factors. Conclusion: There is an independent correlation between sarcopenia and NAFLD in male patients with type 2 diabetes, and sarcopenia may be an independent risk factor for male patients with NAFLD.

Effects of lncRNA HOTAIR on proliferation and apoptosis of myeloma cells through NF-κB pathway.

OBJECTIVE: The aim of this study was to explore the influences of long non-coding ribonucleic acid (lncRNA) homeobox transcript antisense intergenic RNA (HOTAIR) on the proliferation and apoptosis of myeloma cells and its molecular mechanism. MATERIALS AND METHODS: The myeloma cells were randomly divided into three groups, including: group A (myeloma cell group), group B [HOTAIR-small-interfering RNA (siRNA) group], and group C (HOTAIR negative control group). The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed to detect the expression of lncRNA HOTAIR in myeloma cells. The flow cytometry was adopted to determine the apoptosis of myeloma cells. Meanwhile, the protein expression of nuclear factor-kappa-light-chain-enhancer of activated B cells (NF-κB) was detected via Western blotting. In addition, the activity of the myeloma cells was measured using methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The expression of HOTAIR in group A, group B, and group C was (2.19 ± 0.33), (1.37 ± 0.25), and (2.51 ± 0.27), respectively. Compared with group A and group C, the expression of HOTAIR was significantly downregulated in group B, in which the cells interfered with siRNAs. The expression of HOTAIR was significantly higher in group C than group A and group B (p<0.05). HOTAIR expression reached the highest level in group C, followed by group A and group B, respectively. The results of MTT indicated that the activity of the myeloma cells significantly increased at 24 h, 48 h, and 72 h in group C when compared with group A and group B. However, the activity of the myeloma cells was relatively low in group B, showing a slow rising trend. The activity of myeloma cells in group A remarkably increased when compared with group B, but it was lower than group C. Furthermore, the activity of the myeloma cells was higher in group C than group B (p<0.05). Western blotting indicated that the protein expression of NF-κB in group A, group B, and group C was (0.79 ± 0.22), (0.51 ± 0.17), and (0.95 ± 0.31), respectively. Compared with group A and group C, the protein expression of NF-κB was significantly downregulated in group B (the cells interfered with siRNAs) (p<0.05). Meanwhile, the protein expression of NF-κB was markedly higher in group C than in group A and group B (p<0.05). The protein expression of NF-κB was the highest in the cells of group C. Flow cytometry demonstrated that the apoptosis rate in group A, group B, and group C was (9.57 ± 1.71), (20.33 ± 1.63), and (8.74 ± 1.23), respectively. Compared with group A and group C, the apoptosis was significantly elevated in group B, in which the cells interfered with siRNAs (p<0.05). Compared with group A, the number of apoptotic myeloma cells significantly decreased in group C (p<0.05). CONCLUSIONS: LncRNA HOTAIR activates the expression of NF-κB in myeloma cells and promotes the proliferation of myeloma cells.

[Application of the five-level pediatric emergency triage system: a single center study].

Objective: To assess the effectiveness in optimizing resources and shortening critical children's waiting time in pediatric emergency department (PED) with five-level pediatric emergency triage system (PETS). Methods: This retrospective study was conducted in the First Affiliated Hospital of Xiamen University after PETS was applied. The data of patients who visited the pediatric emergency department from January 2015 to December 2017 were collected and analyzed, including age, sex, diseases, visiting time, triage rate and destination. Results: A total of 375 985 patients were included, among whom males were 225 308 (59.9%) and females were 150 677 (40.1%), all younger than 14 years of age. The number of critical cases (level Ⅰ, level Ⅱ and level Ⅲ) was increased from 4 719 (3.7%) in 2015, 12 209 (10.2%) in 2016 to 16 188 (12.7%) in 2017. The number of non-critical patients (level Ⅴ) decreased year by year, as from 98 213 (76.8%) in 2015 to 75 210 (62.6%) in 2016 and 78 857 (61.7%) in 2017. The patients who classified as level Ⅰ or levelⅡaccording to the PETS were seen immediately by physician (n=1855, 0.5%). Overall, 119 738 patients (98.3%) who were classified as level Ⅲ or level Ⅳ could be seen by physician in a timely manner according to triage guidelines, while 2 112 patients (1.7%) could not. The mean waiting time was 9.09 min in level Ⅲ, 17.7 min in level Ⅳ, and 55.76 min in level Ⅴ patients, respectively. The critical cases admitted to the intensive care units were 175 (36.2%) in 2015, 350 (62.8%) in 2016 and 374 (66.2%) in 2017. The etiologies were respiratory diseases (73.3%), gastrointestinal diseases (15.8%) and infectious diseases (3.1%). Conclusion: The application of PETS could optimize emergency resources and shorten the waiting time of critically ill children.

Synergistic cytotoxicity between pentachlorophenol and copper in a bacterial model.

Both pentachlorophenol (PCP) and copper compounds have been widely used as wood preservatives, and are commonly found not only in the area near wood-preserving facilities, but also in body fluids and tissues of people who are not occupationally exposed to them. In this study, we found that exposing bacteria to a combination of PCP and copper at non- or sub-toxic concentrations resulted in enhanced cytotoxic effect in a synergistic mode as indicated by both the inhibition of growth and the lowering of the colony-forming ability. The toxicity of the combination PCP/Cu(II) was relieved by hydrophilic chelating agents, thiol compounds and adventitious proteins, but was markedly potentiated by low levels of the lipophilic metal chelating agents.

Intracellular iron, but not copper, plays a critical role in hydrogen peroxide-induced DNA damage.

The role of intracellular iron, copper, and calcium in hydrogen peroxide-induced DNA damage was investigated using cultured Jurkat cells. The cells were exposed to low rates of continuously generated hydrogen peroxide by the glucose/glucose oxidase system, and the formation of single strand breaks in cellular DNA was evaluated by the sensitive method, single cell gel electrophoresis or "comet" assay. Pre-incubation with the specific ferric ion chelator desferrioxamine (0.1-5.0 mM) inhibited DNA damage in a time- and dose-dependent manner. On the other hand, diethylenetriaminepentaacetic acid (DTPA), a membrane impermeable iron chelator, was ineffective. The lipophilic ferrous ion chelator 1,10-phenanthroline also protected against DNA damage, while its nonchelating isomer 1,7-phenanthroline provided no protection. None of the above iron chelators produced DNA damage by themselves. In contrast, the specific cuprous ion chelator neocuproine (2,9-dimethyl-1,10-phenanthroline), as well as other copper-chelating agents, did not protect against H(2)O(2)-induced cellular DNA damage. In fact, membrane permeable copper-chelating agents induced DNA damage in the absence of H(2)O(2). These results indicate that, under normal conditions, intracellular redox-active iron, but not copper, participates in H(2)O(2)-induced single strand break formation in cellular DNA. Since BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester), an intracellular Ca(2+)-chelator, also protected against H(2)O(2)-induced DNA damage, it is likely that intracellular Ca(2+) changes are involved in this process as well. The exact role of Ca(2+) and its relation to intracellular transition metal ions, in particular iron, needs to be further investigated.

Inhibition of low-density lipoprotein oxidation by carnosine histidine.

Carnosine is a beta-alanylhistidine dipeptide found in skeletal muscle and nervous tissue that has been reported to possess antioxidant activity. Carnosine is a potential dietary antioxidant because it is absorbed into plasma intact. This research investigated the ability of carnosine to inhibit the oxidation of low-density lipoprotein (LDL) in comparison to its constituent amino acid, histidine. Carnosine (3 microM) inhibited Cu2+-promoted LDL (20 of protein/mL) oxidation at carnosine/copper ratios as low as 1:1, as determined by loss of tryptophan fluorescence and formation of conjugated dienes. Carnosine (6 microM) lost its ability to inhibit conjugated diene formation and tryptophan oxidation after 2 and 4 h of incubation, respectively, of LDL with 3 microM Cu2+. Compared to controls, histidine (3 microM) inhibited tryptophan oxidation and conjugated diene formation 36 and 58%, respectively, compared to 21 and 0% for carnosine (3 microM) after 3 h of oxidation. Histidine was more effective at inhibiting copper-promoted formation of carbonyls on bovine serum albumin than carnosine, but carnosine was more effective at inhibiting copper-induced ascorbic acid oxidation than histidine. Neither carnosine nor histidine was a strong inhibitor of 2,2'-azobis(2-amidinopropane) dihydrochloride-promoted oxidation of LDL, indicating that their main antioxidant mechanism is through copper chelation.

The lethal interaction and formation of a lipophilic ternary complex between 2,4,5-trichlorophenol and the Cu(II)-bis(1,10-phenanthroline) complex.

When nonlethal levels of 2,4,5-trichlorophenol (0.2 mM) and the Cu(II)-bis(1,10-phenanthroline) complex [Cu(II)(OP)2] (0.1 microM) were combined, a remarkable synergistic cytotoxicity was observed as measured by the extent of bacterial inactivation. In contrast, no such synergism was observed for the combination of 2,4,5-trichlorophenol with the Cu(II)-bis(bathophenanthroline disulfonate) complex [Cu(II)(BPS)2] which has a chemical structure similar to Cu(II)(OP)2, except for the net charge. The synergism observed for 2,4,5-trichlorophenol and Cu(II)(OP)2 was found to be due to the neutralization of their opposite charge and formation of a lipophilic ternary complex which facilitated copper transport into the bacterial cells.

Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E).

The premise that oxidative stress, among several other factors, plays an important role in atherogenesis implies that the development and progression of atherosclerosis can be inhibited by antioxidants. In this minireview we discuss several mechanisms by which the antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) may protect against atherosclerosis. These mechanisms include inhibition of LDL oxidation and inhibition of leukocyte adhesion to the endothelium and vascular endothelial dysfunction. Overall, ascorbate appears to be more effective than alpha-tocopherol in mitigating these pathophysiological processes, most likely as a result of its abilities to effectively scavenge a wide range of reactive oxygen and nitrogen species and to regenerate alpha-tocopherol, and possibly tetrahydrobiopterin, from its radical species. In contrast, alpha-tocopherol can act either as an antioxidant or a pro-oxidant to inhibit or facilitate, respectively, lipid peroxidation in LDL. However, this pro-oxidant activity of alpha-tocopherol is prevented by ascorbate acting as a coantioxidant. Therefore, an optimum vitamin C intake or body status may help protect against atherosclerosis and its clinical sequelae, whereas vitamin E may only be effective in combination with vitamin C.

Copper-mediated toxicity of 2,4,5-trichlorophenol: biphasic effect of the copper(I)-specific chelator neocuproine.

The lipophilic copper(I)-specific chelator neocuproine has been frequently used as an inhibitor of copper-mediated damage in biological systems. In this communication we report that the copper-mediated toxicity of 2,4,5-trichlorophenol is markedly potentiated by neocuproine at levels which are near-stoichiometric with respect to the copper concentration but is inhibited at higher concentrations. However, no potentiation was observed when neocuproine was substituted by bathocuproinedisulfonic acid, a negative charged ligand with essentially the same copper-binding characteristics as neocuproine. We found that the potentiation by neocuproine was due to the formation of a lipophilic copper complex, while the inhibition by bathocuproinedisulfonic acid was due to the formation of a hydrophilic one. Caution in the use of neocuproine to study copper-mediated toxicity is advised.

Protection by desferrioxamine and other hydroxamic acids against tetrachlorohydroquinone-induced cyto- and genotoxicity in human fibroblasts.

Tetrachlorohydroquinone (TCHQ) has been identified as a major toxic metabolite of the widely used wood preservative pentachlorophenol and has also been implicated in its genotoxicity. We have recently demonstrated that protection by the trihydroxamate iron chelator desferrioxamine (DFO) on TCHQ-induced single-strand breaks in isolated DNA was not the result of its chelation of iron but rather of its efficient scavenging of the reactive tetrachlorosemiquinone (TCSQ) radical. In this study, we extended our research from isolated DNA to human fibroblasts. We found that DFO provided marked protection against both the cyto- and genotoxicity induced by TCHQ in human fibroblasts when it was incubated simultaneously with TCHQ. Pretreatment of the cells with DFO followed by washing also provided marked protection, although less efficiently compared with the simultaneous treatment. Similar patterns of protection were also observed for three other hydroxamic acids (HAs): aceto-, benzo-, and salicylhydroxamic acid. Dimethyl sulfoxide, an efficient hydroxyl radical scavenger, provided only partial protection even at high concentrations. In vitro studies showed that the HAs tested effectively scavenged the reactive TCSQ radical and enhanced the formation of the less reactive and less toxic 2,5-dichloro-3, 6-dihydroxy-1,4-benzoquinone (chloranilic acid). The results of this study demonstrated that the protection provided by DFO and other HAs against TCHQ-induced cyto- and genotoxicity in human fibroblasts is mainly through scavenging of the observed reactive TCSQ radical and not through prevention of the Fenton reaction by the binding of iron in a redox-inactive form.

Evidence for production of hydroxyl radicals by pentachlorophenol metabolites and hydrogen peroxide: A metal-independent organic Fenton reaction.

The production of hydroxyl radicals by tetrachlorohydroquinone (TCHQ, a major metabolite of the widely used biocide pentachlorophenol) in the presence of H(2)O(2) was studied by salicylate hydroxylation method. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl radicals react with salicylate. We found that TCHQ and H(2)O(2) could produce both 2,3- and 2,5-DHBA when incubated with salicylate. Their production was markedly inhibited by hydroxyl radical scavenging agents dimethyl sulfoxide and ethanol, as well as by tetrachlorosemiquinone radical scavengers desferrioxamine and other hydroxamic acids. In contrast, their production was not affected by the nonhydroxamate iron chelators diethylenetriaminepentaacetic acid (DTPA), bathophenanthroline disulfonic acid, and phytic acid, as well as the copper-specific chelator bathocuprione disulfonic acid. A comparison of product formation and distribution from the reaction of ferrous iron with hydrogen peroxide (the classic Fenton system) strongly suggests that the same hydroxyl radical adducts are formed as in the TCHQ/H(2)O(2) experiments. Taken together, we propose that hydroxyl radicals were produced by TCHQ in the presence of H(2)O(2), probably through a metal-independent organic Fenton reaction.

Mechanism of the synergistic cytotoxicity between pentachlorophenol and copper-1,10-phenanthroline complex: the formation of a lipophilic ternary complex.

When non- or sub-toxic levels of pentachlorophenol (PCP) and bis-(1, 10-phenanthroline)cupric complex, Cu(II)(OP)(2), were combined, a remarkable synergistic toxicity was observed as indicated by growth inhibition and bacterial inactivation. Similar synergistic cytotoxic effects were observed with other polychlorinated phenols and other positively charged cupric complexes. The synergism observed for these chemicals and similar reactive pairs of chemicals was found to be due to the formation of lipophilic ternary complexes which facilitated copper transport into the bacterial cells. The formation of ternary complexes of similar lipophilic character could be of relevance as a general mechanism of toxicity.

Application of secondary structure prediction in antisense drug design targeting protein kinase C-alpha mRNA and QSAR analysis.

AIM: To optimize the design of antisense drug targeting protein kinase C-alpha (PKC-alpha) mRNA and obtain better antisense drugs than ISIS3521 that is undergoing clinical trials. METHODS: RNAstructure (version 3.21, 1999) was utilized to predict the optimal and suboptimal secondary structures of human PKC alpha mRNA (GenBank, X52479), and 29 antisense phosphorothioate oligodeoxynucleotides (S-ODN) targeting the secondary structural elements, 3 partly matched S-ODN and 1 scrambled 3521 were designed. ISIS3521 was set as positive control. Mean (n = 3-5) 50% inhibitory effects on proliferation of A549 cells (IC50) of S-ODN were evaluated. Free energies (delta G degree 37) relating to the target secondary structural elements were calculated according to the nearest neighbor model. The quantitative structure-activity relationship (QSAR) analysis through multiple regression was obtained by SPSS. RESULTS: Three S-ODN; (5'-AGCCCA-GCCGCTTGGCTGGG-3', 5'-AGGAGTGCAGCTGC-GTCAAG-3', 5'-TCAGAGGG-ACTGATGACTTT-3') had lower IC50[(48 +/- 7), (50 +/- 4), (64 +/- 2.7) nmol.L-1, respectively] than that of ISIS3521 [(81 +/- 25) nmol.L-1]. The number of bases comprising the target secondary structural element bulge loop, internal loop, and knot, the free energy of S-ODN (delta G degree 37S), and reaction (delta G degree 37R) were important parameters in QSAR equation. In the multiple regression, R was 0.68, P = 0.0193. Not tally with the equation, two S-ODN (5'-TCAAATGGAGG-CTGCCCGGC-3', 5'-AAAACGTCAGCCATGGTCCC-3') with favorable target structures and delta G degree 37 did not behave good activities. CONCLUSION: Computer aided design was helpful to obtain S-ODN with better in vitro effect than current positive drug. The degree of instability of secondary structural elements and delta G degree 37 were important factors for drug activity. Other important factors needed for further investigation.

New modes of action of desferrioxamine: scavenging of semiquinone radical and stimulation of hydrolysis of tetrachlorohydroquinone.

Desferrioxamine (DFO) is a common drug used in the treatment of iron overload. In addition to its iron-chelation, other properties have been identified. Alas, DFO has demonstrable effects which cannot be explained by its classically established properties; i.e., DFO protects against DNA single strand breaks induced by tetrachlorohydroquinone (TCHQ), while other iron chelators such as DTPA (diethylenetriaminepentaacetic acid) do not. The autooxidation process of TCHQ yielding the tetrachlorosemiquinone radical (TCSQ.) intermediate, was studied here in the presence of chelators. DFO led to a marked reduction in both concentration and life span of TCSQ. via formation of DFO-nitroxide radical (DFO.). In contrast, DTPA had no detectable effect on TCHQ autooxidation. Present studies indicate that the protective effects of DFO on TCHQ-induced DNA damage were not due to the binding of iron, but rather to scavenging of the reactive TCSQ. and the formation of the less reactive DFO.. An additional mode of action of DFO was identified, via stimulation of the hydrolysis (dechlorination) of tetrachloro-1,4-benzoquinone (chloranil), which is the oxidation product of TCHQ, to form 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (chloranilic acid). The results of this study demonstrate two new modes of action for DFO: the scavenging of deleterious semiquinone radical, and the stimulation of the hydrolysis of halogenated substituents on the quinone structure. Both modes might prove highly relevant to the biological activities of DFO.