New insight into the negative impact of imidazolium-based ionic liquid [C10mim]Cl on Hela cells: From membrane damage to biochemical alterations.

As an alternative to volatile organic solvents, ionic liquids (ILs) are known as "green solvents", and widely used in industrial applications. However, due to their high solubility and stability, ILs have tendency to persist in the water environment, thus having potential negative impacts on the aquatic ecosystem. For assessing the environmental risks of ILs, a fundamental understanding of the toxic effects and mechanisms of ILs is needed. Here we evaluated the cytotoxicity of 1-methyl-3-decylimidazolium chloride ([C10mim]Cl) and elucidated the main toxic mechanism of [C10mim]Cl in human cervical carcinoma (Hela) cells. Microstructural analysis revealed that [C10mim]Cl exposure caused the cell membrane breakage, swollen and vacuolated mitochondria, and spherical cytoskeletal structure. Cytotoxicity assays found that [C10mim]Cl exposure increased ROS production, decreased mitochondrial membrane potential, induced cell apoptosis and cell cycle arrest. These results indicated that [C10mim]Cl could induce damage to cellular membrane structure, affect the integrity of cell ultrastructure, cause the oxidative damage and ultimately lead to the inhibition of cell proliferation. Moreover, alterations of biochemical information including the increased ratios of unsaturated fatty acid and carbonyl groups to lipid, and lipid to protein, and the decreased ratios of Amide I to Amide II, and α-helix to ß-sheet were observed in [C10mim]Cl treated cells, suggesting that [C10mim]Cl could affect the structure of membrane lipid alkyl chain and cell membrane fluidity, promote the lipid peroxidation and alter the protein secondary structure. The findings from this work demonstrated that membrane structure is the key target, and membrane damage is involved in [C10mim]Cl induced cytotoxicity.

Microbial transformation of progesterone and dydrogesterone by bacteria from swine wastewater: Degradation kinetics and products identification.

Natural and synthetic progestogens in livestock environments have become a concern due to the frequent presence and potential adverse effects on aquatic organisms. Here we investigated the biotransformation of progestogens by wastewater-borne bacteria in the field and laboratory under oxic and anoxic conditions. The results showed that all progestogens dissipated faster under oxic conditions than under anoxic conditions, and natural progesterone transformed faster than synthetic progestogens. Meanwhile, dozens of bacterial strains capable of degrading progestogens were successfully isolated from the swine wastewater, and Bacillus sp. P19 and Bacillus sp. DGT2 were found the best for progesterone and dydrogesterone transformation, respectively. In the degradation experiments using a single bacterial strain, progesterone and dydrogesterone dissipated under oxic conditions with half-lives of 11.6 h and 18.2 h, respectively. The transformation pathways were proposed based on the identified transformation products. The findings from this study showed that progestogens can be biotransformed, but not fully mineralized in the environment.

Occurrence, toxicity and transformation of six typical benzotriazoles in the environment: A review.

Benzotriazoles (BTs) are a group of heterocyclic compounds which have been widely applied in industrial activities and domestic life mainly as corrosive inhibitors. BTs have been ubiquitously detected in receiving environments and cause potential toxicity to non-target organisms. This paper reviews the occurrence and fate of six selected benzotriazole compounds in different environmental and biological matrices, as well as the transformation and toxicity. Due to their high hydrophilicity and insufficient removal in wastewater treatment plants (WWTPs), these compounds were widely detected in aquatic environments with concentrations mainly from tens ng/L to tens µg/L. Considerable residual levels of BTs in plant, fish, air, tap water and human urine have implied the potential risks to various organsims. The reported acute toxicity of BTs are generally low (EC50 in mg/L level). Some observed sublethal effects including endocrine disrupting effects, hepatotoxicity and neurotoxicity, as well as the ability to promote the development of endometrial carcinoma still raise a concern. BTs are found often more recalcitrant to biodegradation compared to photolysis and ozonation. Environmental factors including pH, temperature, irradiation wavelength, redox condition as well as components of matrix are proved crucial to the removal of BTs. Further studies are needed to explore the precise environment fate and toxicity mechanism of BTs, and develop advanced treatment technologies to reduce the potential ecological risks of BTs.

Dydrogesterone affects the transcription of genes in GnRH and steroidogenesis pathways and increases the frequency of atretic follicles in zebrafish (Danio rerio).

Dydrogesterone (DDG) is a synthetic progestin broadly used in human and veterinary medicine and has been widely detected in aquatic environments. However, its potential effects on aquatic organisms are little documented. Here we investigate the short-term effects of DDG on the transcriptional and histological responses in adult zebrafish (Danio rerio). Adult zebrafish were exposed to 32.0, 305 and 2490 ng L-1 of DDG for 14 days. Real time quantitative PCR analysis showed that DDG significantly increased transcripts of most genes involved in the gonadotropin-releasing hormone (GnRH) pathway in the brain of female. In contrast, apparent down-regulation of these gene transcriptions was observed in the brain of males. The transcription of cyp19a1a in the ovary had a 2.3 fold increase at 2490 ng L-1 of DDG and the transcription of hsd17b2 at 305 and 2490 ng L-1 in the testis was enhanced by approximately 2.0 fold and 2.4 fold, respectively. Histopathological analysis revealed exposure to 2490 ng L-1 DDG significantly increased the percentage of atretic follicles in the ovary. The results of this study suggest that DDG has potential endocrine disrupting effects and affects the ovarian development in zebrafish.

Swine farm wastewater discharge causes masculinization of western mosquitofish (Gambusia affinis).

Natural and synthetic steroid hormones have been detected in swine farm wastewaters (SFWs) and receiving waters. However, little is known on their potential endocrine disrupting effects on fish population in receiving rivers. Here we investigated the concentrations of androgens and estrogens in the SFWs and the receiving waters. In addition, the endocrine disrupting effects of SFWs on wild western mosquitofish (Gambusia affinis) were also analyzed by assessing the sex ratio, secondary sex characteristics and transcriptional expression of genes related to the endocrine system. Chemical analysis showed the maximum concentration of total androgens reached 1375.7 ng L-1 in the SFWs and 1020.8 ng L-1 in receiving river waters. A total of 3552 adult G. affinis were collected from the Lianhe River (Guangdong, China), which was heavily impacted by SFWs, before and after the Ban of Swine Farming (BSF) in the river basin. The results showed the presence of male-biased G. affinis populations and masculinized anal fins and hemal spines of females at all contaminated sites prior to BSF. In addition, the transcription of vitellogenin (vtg) was inhibited by SFWs by 99.9% in the females from one sampling site. In contrast, the adverse effects on the females were significantly decreased after BSF. Redundant and path analyses showed that skewed sex ratios of fish populations, masculinized secondary sex characteristics of females and the decrease of vtg transcripts of females were well correlated with the concentrations of androgens in the receiving waters. The findings from this study suggest that SFW can cause masculinization effects of wild G. affinis populations.

Determination of 24 personal care products in fish bile using hybrid solvent precipitation and dispersive solid phase extraction cleanup with ultrahigh performance liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry.

Personal care products (PCPs) are ubiquitous in aquatic environments owing to the continuous discharge of domestic wastewater from highly urbanized regions. These PCPs can be adsorbed by fish and thereafter usually enter the bile of the fish through biliary excretion. In this study, a sensitive method based on a combination of hybrid solvent precipitation and dispersive solid phase extraction (d-SPE) purification was developed to simultaneously extract and detect 24 PCPs, namely, 16 biocides, 4 synthetic musks, and 4 benzotriazoles, from fish bile. Hybrid precipitation on solid phase extraction (SPE) tubes was applied to remove phospholipids and proteins, and a d-SPE procedure was used for further purification. The extraction solvents for the hybrid precipitation/SPE tubes and d-SPE materials were optimized. The method performance for bile samples both with and without enzyme hydrolysis using ß-glucuronidase/aryl-sulfatase were validated. The 24 PCPs in fish bile were spiked with standard concentrations of 10 ng/mL, 20 ng/mL, 100 ng/mL, and 200 ng/mL to evaluate recoveries, which ranged from 70 to 120% for 16, 16, 22, and 21 analytes with hydrolysis, respectively, and 70-120% for 14, 15, 23, and 23 analytes without hydrolysis, respectively. The quantification limits for target PCPs were in the range 0.26-7.38 ng/mL [excluding musk xylene (MX) and musk ketone (MK)] and 0.20-9.48 ng/mL (excluding MX and MK) for bile samples with and without enzyme hydrolysis, respectively. After enzyme hydrolysis, 12 PCPs were detected in bile from fish collected from the Yangtze River, with a maximum detected concentration of 460 ng/mL, for triclosan (TCS). The hydrolysis reaction indicated that high percentages of glucuronide and sulfate metabolites for some PCPs, i.e. four parabens and TCS, existed in the bile.

Simultaneous determination of 24 personal care products in fish muscle and liver tissues using QuEChERS extraction coupled with ultra pressure liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometer analyses.

A sensitive and selective quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction combined with dispersive solid-phase extraction (d-SPE) cleanup method was developed to simultaneously extract a wide range of personal care products (16 biocides, 4 synthetic musks, and 4 benzotriazoles) in fish muscle and liver tissues. In order to get satisfactory recoveries, different extraction parameters were optimized, including extraction salts and d-SPE materials, extraction solvents and acetic acid contents in organic phase, and the ratios of solvent and water. Ultra pressure liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry were used to analyze the target compounds in the extracts. Among the 24 personal care products, the recoveries in the range of 70-120 % were obtained for 20, 19, and 12 analytes in fish muscle at the spiking concentrations of 10, 5, and 1 ng/g ww, respectively, and for 13, 12, and 11 analytes in liver at the spiking concentrations of 40, 20, and 4 ng/g ww, respectively. Method quantification limits (MQLs) of all analytes were 0.02-2.12 ng/g ww for fish muscle and 0.22-12.2 ng/g ww for fish liver tissues. The method was successfully applied to wild fish samples collected from Dongjiang River, south China. Twenty-one and 17 of the analytes were found in fish muscle and liver samples, respectively, in at least one site of the river with the concentrations between below MQLs and 119 ng/g ww, respectively. Graphical abstract Achieved satisfactory recoveries, high precision, and low method quantification limits (MQLs) for PCPs in wild fish tissues by QuEChERS procedure optimization combined with UPLC-MS/MS and GC-MS analyses.

Evaluation of estrogenic activity in the Pearl River by using effect-directed analysis.

This study investigated estrogenic activity of water, sediment, and fish bile of the Pearl River in southern China by effect-directed analysis based on in vitro yeast screen assay and chemical analysis. Results showed higher estradiol equivalents (EEQ) for surface water in dry season than in wet season. Simple risk assessment suggested that high estrogenic risk would be expected in Shima River and Danshui River receiving discharge of effluents from cities in the region. Fractionation and effect-directed analysis showed that estrogenic activity mainly occurred in relatively polar fractions of surface water. Seven target estrogenic compounds (bisphenol A, 4-nonylphenol, 4-tert-octylphenol, 17α-ethynyl estradiol, estrone, diethylstilbestrol, and 17ß-estradiol) only accounted for part of the measured estrogenic activity, with the rest contributions from other potential estrogenic chemicals such as phenols. Findings from this study suggest that fish in the river could be affected by those estrogenic chemicals. Proper measures should be taken to reduce the estrogenic activity in wastewaters before they are discharged into the riverine system in order to protect aquatic organisms.

Feminization and masculinization of western mosquitofish (Gambusia affinis) observed in rivers impacted by municipal wastewaters.

Municipal wastewaters have been known to contain various estrogens and androgens. Little is known about the joint action of these chemicals from wastewaters on fishes in the aquatic environment. The objectives of this study were to investigate the estrogenic and/or androgenic effects in wild mosquitofish (Gambusia affinis) of two effluent-impacted rivers in South China by determining morphological changes and hepatic mRNA expression levels of relevant genes such as vitellogenin (Vtg), estrogen receptor (ERα) and androgen receptors (ARα and ARß), and to assess the linkages of those morphological changes and hepatic mRNA expression levels to the chemical concentrations measured by in vitro bioassays and chemical analysis. The results showed a significant induction of Vtg and ERα mRNA in the livers of the males and a gonopodium-like anal fin in the females collected at the majority of sites. Redundancy analysis and Pearson correlation analysis showed that the chemical concentrations obtained by in vitro bioassays and chemical analysis had significant correlations with some of the endpoints for the estrogenic and/or androgenic effects in mosquitofish. The findings from this study indicate that the estrogens and androgens present in the two rivers could cause the observed estrogenic and androgenic effects in mosquitofish.

Biotransformation of progesterone and norgestrel by two freshwater microalgae (Scenedesmus obliquus and Chlorella pyrenoidosa): transformation kinetics and products identification.

Natural and synthetic steroid hormones such as progesterone and norgestrel in the aquatic environment may cause adverse effects on aquatic organisms. This study investigated the biotransformation of progesterone and norgestrel in aqueous solutions by two freshwater microalgae Scenedesmus obliquus and Chlorella pyrenoidosa and elucidated their transformation mechanisms. More than 95% of progesterone was transformed by the two microalgae within 5d. For norgestrel, almost complete transformation by S. obliquus was observed after 5 d, but nearly 40% was remained when incubated with C. pyrenoidosa. The results also showed that these two compounds were not accumulated in the algal cells. Biotransformation was found to be the main mechanism for their loss in the aqueous solutions, and it followed the first-order kinetic model. For progesterone, three main transformation products, i.e. 3ß-hydroxy-5α-pregnan-20-one, 3,20-allopregnanedione and 1,4-pregnadiene-3,20-dione, and six minor androgens were identified. For norgestrel, only two transformation products, 4,5-dihydronorgestrel and 6,7-dehydronorgestrel, were identified for the first time. Hydroxylation, reduction and oxidation are proposed to be the main transformation pathways. Among the two microalgae species, S. obliquus was found more efficient in the transformation of the two target compounds than C. pyrenoidosa. The results clearly demonstrated the capability of the two microalgae to transform the two progestogens. The biotransformation and products could have significant environmental implications in the fate and effects of the two steroids.

Degradation of norgestrel by bacteria from activated sludge: comparison to progesterone.

Natural and synthetic progestagens in the environment have become a concern due to their adverse effects on aquatic organisms. Laboratory studies were performed to investigate aerobic biodegradation of norgestrel by bacteria from activated sludge in comparison with progesterone, and to identify their degradation products and biotransformation pathways. The degradation of norgestrel followed first order reaction kinetics (T1/2 = 12.5 d), while progesterone followed zero order reaction kinetics (T1/2 = 4.3 h). Four and eight degradation products were identified for norgestrel and progesterone, respectively. Six norgestrel-degrading bacterial strains (Enterobacter ludwigii, Aeromonas hydrophila subsp. dhakensis, Pseudomonas monteilii, Comamonas testosteroni, Exiguobacterium acetylicum, and Chryseobacterium indologenes) and one progesterone-degrading bacterial strain (Comamonas testosteroni) were successfully isolated from the enrichment culture inoculated with aerobic activated sludge. To our best knowledge, this is the first report on the biodegradation products and degrading bacteria for norgestrel under aerobic conditions.

Effects of steroid hormones on reproduction- and detoxification-related gene expression in adult male mosquitofish, Gambusia affinis.

The molecular mechanisms that mediate fish reproduction and detoxification in response to steroid hormones were studied by using adult male western mosquitofish (Gambusia affinis) as sentinel species. The expression patterns of three vitellogenins (VtgA, VtgB and VtgC), two estrogen receptors (ERα and ERß), two androgen receptors (ARα and ARß), metallothionein (MT) and cytochrome P450 1A (CYP1A) in the liver and testis of adult male mosquitofish were assessed through exposure treatments with progesterone (P), testosterone (T) and 17ß-estradiol (E2), alone and in combination for eight days. The results showed that expression patterns of Vtg subtype, ER subtype, AR subtype, MT and CYP1A genes in male mosquitofish varied according to tissue and specific hormone stress. Vtg subtype mRNA expression was induced in the liver in E2-added treatments, and an up-regulation of ERα mRNA expression was also observed. In addition, hormone treatments increased three Vtg subtype mRNA expression levels in the testis, at least to some extent. All hormone treatments significantly inhibited ERα, ERß and ARß mRNA expression in the testis. Some of hormone treatments could affect MT and CYP1A gene expression in mosquitofish. In general, multiple hormone treatments showed different effects on target gene expression compared with corresponding hormone alone. The results from the present study provided valuable information on the toxicological effects of steroid hormones in mosquitofish.

Hormonal effects of tetrabromobisphenol A using a combination of in vitro and in vivo assays.

The aim of this study was to investigate the hormonal effects of tetrabromobisphenol A (TBBPA) in vitro on recombinant yeasts and in vivo on mosquitofish (Gambusia affinis). The in vitro bioassays for (anti-)androgenic activities showed that TBBPA had a weak androgenic activity in vitro with recombinant yeast systems carrying human androgen receptor (hAR). In the in vivo bioassays, the gene expression patterns of vitellogenin (Vtg), estrogen receptors (ERα and ERß), and androgen receptors (ARα and ARß) in adult males and juveniles after exposure to TBBPA for 60 days were evaluated. Significant up-regulation of Vtg, ERα, and ERß mRNAs was observed in the liver after exposure to 500 nM of TBBPA. In the testis, the lowest concentration of TBBPA (50 nM) markedly induced Vtg, ERß, and ARß mRNA expression, but the same concentration significantly inhibited ARα mRNA expression. In addition, in juveniles, 100 nM of TBBPA significantly up-regulated the expression of Vtg, ERß, and ARα mRNAs. However, TBPPA did not cause histological alterations in the liver and testis of adult male mosquitofish. The results from this present study suggest that TBBPA could display low but multiple hormonal activities despite its low toxicity to mosquitofish.

Simultaneous determination of benzotriazoles and ultraviolet filters in ground water, effluent and biosolid samples using gas chromatography-tandem mass spectrometry.

A new method using gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for the determination of four benzotriazoles, i.e. benzotriazole (BT), 5-methylbenzotriazole (5-TTri), 5-chlorobenzotriazole (CBT), 5,6-dimethylbenzotriazole (XTri), and six UV filters, i.e. benzophenone-3 (BP-3), 3-(4-methylbenzylidene)camphor (4-MBC), octyl 4-methoxycinnamate (OMC), 2-(3-t-butyl-2-hydroxy-5-methylphenyl)-5-chloro benzotriazole (UV-326), 2-(2'-hydroxy-5'-octylphenyl)-benzotriazole (UV-329), and octocrylene (OC) in ground water, effluent and biosolid samples. Solid phase extraction (SPE) and pressurized liquid extraction (PLE) were applied as the preconcentration method for water samples (ground water and effluent) and biosolid samples, respectively. The optimized method allowed us to quantify all target compounds with the method detection limits ranging from 0.29 to 11.02 ng/L, 0.5 to 14.1 ng/L and 0.33 to 8.23 ng/g in tap water, effluent and biosolid samples, respectively. The recoveries of the target analytes in tap water, effluent and biosolid samples were 70-150%, 82-127% and 81-133%, respectively. The developed analytical method was applied in the determination of these target compounds in ground water, effluent and biosolid samples collected from Bolivar sewage treatment plants in South Australia. In effluent samples, the target compounds BT, 5-TTri, CBT, XTri and BP-3 tested were detected with the maximum concentration up to 2.2 µg/L for BT. In biosolid samples, eight out of ten compounds tested were found to be present at the concentrations ranging between 18.7 ng/g (5-TTri) and 250 ng/g (4-MBC).

LBP and CD14 polymorphisms correlate with increased colorectal carcinoma risk in Han Chinese.

AIM: To explore the associations of polymorphisms of lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) with the colorectal carcinoma (CRC) risk in Han Chinese. METHODS: Polymorphisms of LBP (rs1739654, rs2232596, rs2232618), CD14 (rs77083413, rs4914), TLR-4 (rs5030719), IL-6 (rs13306435) and TNF-α (rs35131721) were genotyped in 479 cases of sporadic colorectal carcinoma and 486 healthy controls of Han Chinese in a case-control study. Single-nucleotide polymorphisms (SNPs) between cases and controls were analyzed by unconditional logistic regression. RESULTS: GA and GG genotypes of LBP rs2232596 were associated with a significantly increased risk of CRC [odds ratio (OR) = 1.51, 95% confidence interval (CI) 1.15-1.99, P = 0.003; OR = 2.49, 95% CI 1.16-5.38, P = 0.016, respectively]. A similar association was also observed for the CG genotype of CD14 rs4914 (OR= 1.69, 95% CI 1.20-2.36, P = 0.002). In addition, a combination of polymorphisms in LBP rs2232596 and CD14 rs4914 led to a 3.4-fold increased risk of CRC (OR = 3.44, 95% CI 1.94-6.10, P = 0.000). CONCLUSION: This study highlights the LBP rs2232596 and CD14 rs4914 polymorphisms as biomarkers for elevated CRC susceptibility in the Chinese Han population.

Estrogenic activity profiles and risks in surface waters and sediments of the Pearl River system in South China assessed by chemical analysis and in vitro bioassay.

Estrogenic activity risks in the Pearl River system (Liuxi River, Zhujiang River and Shijing River) in South China were assessed by combined chemical analysis and recombinant yeast estrogen screen (YES) bioassay for surface waters and sediments collected in both dry and wet seasons. The xenoestrogens 4-tert-octylphenol, 4-nonylphenol and bisphenol A were detected at almost every sampling site at concentrations of several ng L(-1) (ng g(-1)) to tens of µg L(-1) (µg g(-1)) in surface waters (and sediments). The estrogens estrone and 17ß-estradiol were also detected in most of the samples with concentrations from several ng L(-1) (ng g(-1)) to tens of ng L(-1) (ng g(-1)) in surface waters (and sediments). However, synthetic estrogens diethylstilbestrol and 17α-ethinylestradiol were only detected at a few sites. The 17ß-estradiol equivalents (EEQ) screened by the YES bioassay were in the range of 0.23-324 ng L(-1) in surface waters and from not detected to 101 ng g(-1) in sediments. Shijing River displayed one to two orders of magnitude higher levels for both measured chemical concentrations and estrogenic activities than the Zhujiang River and the Liuxi River. A risk assessment for the surface waters showed high risks for the downstream reaches of the Liuxi River and the upstream to midstream reaches of the Zhujiang River and the Shijing River. Higher estrogenic risks were observed in the wet season than in the dry season for surface waters, probably due to the input of runoff and direct overflow of small urban streams during heavy rain events. Only small variations in estrogenic risk were found for the sediments between the two seasons, suggesting that sediments are a sink for these estrogenic compounds in the rivers.

The localization of two mimic epitopes of lipopolysaccharide binding protein and the preparation of their tandem multiple antigen peptide.

OBJECTIVE: To screen and identify two mimic epitopes in the inflammatory site of lipopolysaccharide binding protein (LBP), and to synthesize and purify their corresponding mimic epitope four branched peptide (multiple antigen peptide, MAP). METHOD: Using an anti-full length LBP monoclonal antibody as the target molecule, the amino acid sequences of the exogenous peptides were deduced by combining several different techniques including: affinity screening of the phage display peptide library, the lipopolysaccharide (LPS) binding activity assay and competitive inhibition test, cytokine production inhibition, flow cytometry, and DNA sequencing. Basic Local Alignment Search Tool (BLAST) software was used to compare the resulting peptide sequences with the primary structure sequence of the LBP molecule, and thus the amino acid sequences for two mimic inflammatory epitopes for the binding of LBP and LPS were determined. Additionally, the two target sequences were coupled, and the 9-fluorenylmethyloxycarbonyl (FMOC) solid-phase synthesis method was used to synthesize the 24aa peptide. The design program of the multiple antigen peptide (MAP) was used to couple the four tandem peptides with lysine as the core base to produce the branch like structure, and thus, the four branched peptide was synthesized and purified. RESULTS: Fourteen phage clones (C) with competitive LPS binding activity with LBP were successfully obtained. Among these, the amino acid sequences of the peptides in C2, C19, C57, C77, C85 and C91 showed a homology of more than 90% to the primary structure of LBP. However, the amino acid sequences of C29 and C90, WKAQKRFMKKSG and LKTRKLFWKYKD, respectively, did not show homology to the primary structure of LBP, which were determined to be mimic epitopes of the inflammatory sites in LBP. Further synthesis of the 24aa peptide using FMOC solid-phase synthesis and MAP modification were carried out, the four branched peptide was synthesized and purified, and the purity was found to be higher than 95%. The purified peptide was subjected to mass spectrometry analysis and amino acid analysis, and its molecular weight (3102.77 kDa) and amino acid composition were in accordance with theoretical values. CONCLUSION: The amino acid sequence for two mimic epitopes of the inflammatory site of LBP were determined to be WKAQKRFMKKSG and LKTRKLFWKYKD. The MAP was successfully prepared simultaneously and is able to be used as the core antigen protein for the formulation of vaccines. This knowledge will help in future investigations of the functional characteristics of LBP protein, and enhance exploration into new pathways for the prevention and treatment of LPS inflammatory diseases.

A synthetic MD-2 mimetic peptide attenuates lipopolysaccharide-induced inflammatory responses in vivo and in vitro.

Myeloid differentiation protein-2 (MD-2), a secreted glycoprotein that binds to both lipopolysaccharide (LPS) and toll like receptor 4 (TLR4), contributes to the fine ligand recognition and signaling activation on LPS-induced inflammation. Here we synthesized a novel MD-2 mimetic peptide (MDMP), derived from the putative LPS-binding domain and TLR4-binding domain of MD-2, and found that MDMP dose-dependently bound to LPS and inhibited LPS-activated Limulus amebocyte lysate (LAL). Pretreatment with MDMP dampened LPS-induced inflammatory responses in RAW264.7 cells, including down-regulation of TLR4-MD-2 complex on the cell surface, suppression of LPS binding to the cells, inhibition of mitogen-activated protein kinase (MAPKs) and nuclear factor kappa B (NF-kappaB) activation, reduction of tumor necrosis factor-alpha (TNF-alpha) production. Further, in vivo pretreatment with MDMP markedly protected against LPS-induced acute lung injury and liver injury, as indicated by the notable reduction of lethality, inflammatory responses and TNF-alpha production. These results demonstrate that MDMP attenuates LPS-induced inflammatory responses in vivo and in vitro, and suggests that MDMP may be useful in the treatment of inflammation associated with LPS.

Analysis of the early adaptive response of endothelial cells to hypoxia via a long serial analysis of gene expression.

Activation of endothelial cells in humans is an early event in the response to hypoxia that may contribute to the endothelium's endogenous capacity to reduce tissue injury. To better understand the mechanism underlying this process, we utilized Long Serial Analysis of Gene Expression to study the transcriptome of human vein umbilical endothelial cells (EA.hy926) shortly after the induction of hypoxia. Of over 13,000 genes detected in each pool, 112 showed obvious differences in expression. Metabolic processes such as protein biosynthesis and proteolysis, aminoglycan metabolism, ribonucleotide biosynthesis, adenosine salvage, and lipid metabolism were reinforced. Pro-proliferation and pro-apoptotic states suggest the co-existence of pro- and anti-injury forces in endothelium shortly after the induction of hypoxia. Other adaptive responses include reinforced angiogenesis and vasodilation. Additionally, gene transcription in the endothelium shortly after the induction of hypoxia was regulated independently of HIF-1alpha. Our efforts to elucidate the adaptive response at an early post-hypoxia stage should contribute to further investigation of the protective processes that occur in the endothelium and has potential clinical implications.

[Clinical related factors of portal vein tumor thrombosis in patients with hepatocellular carcinoma: a logistic regression analysis].

OBJECTIVE: To investigate the related factors of portal vein tumor thrombosis (PVTT) in patients with HCC. METHODS: A total number of 234 patients with hepatocellular carcinoma (HCC) were included in this retrospective study. Uni-variate and multi-variate logistic regression analysis were employed to analyze the association between PVTT and 18 routine clinical parameters. RESULTS: Among the 234 patients with HCC, 15% of patients (35/235) had PVTT. Univariate logistic regression analysis revealed significant association of age (P = 0.016), gamma glutamyl transferase (GGT, P = 0.003), number of segmental invasion (P = 0.007), microvascular invasion (P < 0.01), segment location of S2 (P = 0.001), S3 (P = 0.000), S4 (P = 0.004) and S6 (P = 0.016). Multivariate analysis shows potential significant predictors of PVTT in HCC were age (RR: 0.373; 95% CI: 0.146-0.954; P = 0.040), the tumor location of S3 (RR: 4.625; 95% CI: 1.916-11. 165;P = 0.001), GGT (RR: 4.091; 95% CI: 1.448-11.553; P = 0.008) and microvascular invasion (RR: 20.912; 95% CI: 4.745-92.172; P < 0.01). CONCLUSIONS: PVTT occurred more commonly in the younger (< 50 years old), and those with high level of GGT, segment location of S3 and microvascular invasion.