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The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is widely used as a cellulase producer in the industry. Herein, we describe the rational engineering of the publicly available T. reesei QM9414 strain to achieve a remarkable high-level production of cellulase on glucose. Overexpression of the key cellulase regulator XYR1 by the copper-repressible promoter Ptcu1 was first implemented to achieve a full cellulase production in the context of catabolite repression (CCR) while eliminating the requirement of inducing sugars for enzyme production. The T. reesei bgl1 gene was further overexpressed to compensate for its low β-glucosidase activity on glucose. This overexpression resulted in a 102% increase in FPase activity compared with the CCR-released RUT-C30 strain cultured on Avicel. Moreover, the saccharification efficiency toward pretreated corncob residues by crude enzymes from the engineered strain on glucose increased by 85% compared with that treated by enzymes from RUT-C30 cultivated on Avicel. The engineered T. reesei strain thus shows great potential as a viable alternative to deliver commercial cellulases after further optimization for efficient saccharification of agricultural waste.

Enzymatic hydrolysis of xylan represents a promising way to produce xylooligosaccharide (XOS), which is a novel ingredient in functional food. However, the recalcitrance of xylan in natural lignocellulosic biomass entails effective and robust xylanases. In the present study, we reported the isolation of a thermophilic Streptomyces sp. B6 from mushroom compost producing high xylanase activity. Two xylanases of Streptomyces sp. B6 belonging to GH10 (XynST10) and GH11 (XynST11) families were thus identified and biochemically characterized to be robust enzymes with high alkaline- and thermostability. Direct hydrolysis of neutralized viscose fiber production waste using XynST10 and XynST11 showed that while XynST10 produced 23.22 g/L XOS with a degree of polymerization (DP) of 2-4 and 9.27 g/L xylose, XynST11 produced much less xylose (1.19 g/L) and a higher amounts of XOS with a DP = 2-4 (28.29 g/L). Thus, XynST11 holds great potential for the production of XOS from agricultural and industrial waste.

Nucleosome binding protein 1 (NSBP1) is identified as a new member of HMGN family and is abnormally overexpressed in a variety of tumors. However, it remains unclear whether NSBP1 is overexpressed and promotes gastric cancer. In this study we employed RNAi mediated knockdown of NSBP1 to investigate potential oncogenic role of NSBP1 in gastric cancer. In BGC823 and SGC7901 gastric cancer cell lines, we showed that NSBP1 knockdown decreased cell proliferation while increased apoptosis in vitro. Western blot analysis showed that NSBP1 knockdown decreased the levels of anti-apoptotic protein Bcl-2 while increased the levels of pro-apoptotic protein Bax. In addition, NSBP1 knockdown inhibited the growth and increased the apoptosis of SGC7901 cells xenografted in nude mice. In conclusion, this study provides the first evidence that NSBP1 enhances the proliferation while inhibits the apoptosis of gastric cancer cells, and this is related to the regulation of the expression of apoptosis related proteins by NSBP1. These data suggest that NSBP1 plays oncogenic role in gastric cancer.

Background Myocardial ischemia (MI) often causes angina, arrhythmia, and cardiac insufficiency, sometimes resulting in death. Ischemia-induced myocardial tissue damage is attributed to the hypoxic damage of myocardial cells producing apoptosis and decreased proliferation. Taurine has been shown to improve MI, but its mechanism is largely unknown. Methods In this study, the relationship between taurine and severity of MI in vivo was evaluated by quantifying myocardial infarct areas and metabolic indicators of myocardial damage and measuring taurine levels in cardiac muscle and plasma by high performance liquid chromatography (HPLC). To elucidate how taurine might suppress ischemic injury, we established an in vitro ischemia model with isolated primary rat cardiomyocytes cultured without serum or glucose and under hypoxia. We evaluated the indicators of MI and damage, including lactic dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI). We also examined the levels of taurine transporter (TauT), cysteine dioxygenase (CDO), and cysteine sulfinate decarboxylase (CSD) proteins involved in transport and synthesis of taurine in the myocardium and those of 2 apoptosis-associated proteins, namely, Bcl-2 associated X protein (BAX) and B-cell lymphoma-2 (Bcl-2). Results Exposure of myocardial cells to ischemia led to the decrease of taurine content, the suppression of cell proliferation, and led to calcium ion overload and apoptosis. Pretreatment with taurine alleviated the ischemic damage, with concomitant elevation of intracellular taurine concentrations. Molecular mechanism analysis showed that pretreatment with taurine upregulated the TauT, CDO, and CSD, 2 rate-limiting enzymes involved in taurine synthesis. These effects facilitated both taurine transport into cells and taurine synthesis, leading to taurine accumulation. In addition, apoptosis inhibition by taurine appeared to be mediated by upregulated Bcl-2 and downregulated BAX, as well as inhibition of calcium overload by suppression of calcium binding protein. Conclusions We demonstrated that TauT is critical for the attenuation of myocardial ischemic damage by taurine, facilitating taurine absorption and synthesis. These findings provided new insights and a theoretical foundation for future studies examining taurine as a potential treatment for MI.

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei., Author summary Originally identified in budding yeast, CYC8 (also known as SSN6)/TUP1 (also known as RCOA/RCO1) is one of the best studied corepressors and has served as a model for the study of similar corepressor complexes in higher eukaryotes. Besides its well-established role in transcriptional repression, CYC8/TUP1 has been reported to be involved in transcriptional activation following release from repression in S. cerevisiae. In filamentous fungi, there have been also reports on TUP1 homolog functions in specific gene repression as well as vegetative growth and asexual spore production. However, CYC8/TUP1 function in gene activation has not been described. In this study, we reveal a coactivator function for TrCYC8/TUP1 in initiating the cellulolytic response in T. reesei. TrCYC8/TUP1 itself is specifically recruited to cellulase gene promoters in an XYR1-dependent manner. We further show that TrCYC8/TUP1 contributes to the induced cellulase gene expression by improving the binding of the essential transcriptional activator XYR1. These results thus implicate a synergistic mechanism in ensuring the efficient recruitment of XYR1 and TrCYC8/TUP1 to target gene promoters.

EGCG is a major pharmacological compound in green tea. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. Inflammation and insulin resistance are involved in the development of the disease. In this study, we investigated the beneficial effect of EGCG on the liver tissue of NAFLD rats induced by a high-fat diet and its underlying mechanism. Thirty Sprague-Dawley rats received a normal diet, a HFD and a HFD+EGCG. The expression levels of inflammatory signaling pathway genes (e.g., TLR4, TRAF6, IKKβ, NF-κB, TNF-α) and insulin signaling transduction pathway genes (e.g., PI3K, AKT, IRS-1, IRS-2) were detected in the liver. We observed that EGCG decreased the triglyceride (TG) concentration in rat livers and suppressed TLR4, TRAF6, IKKβ, p-IKKβ, p-NF-κB, and TNF-α levels compared with those in the HFD group, whereas PI3K, AKT, IRS-1, and IRS-2 indicators were improved. EGCG improves obesity-associated subacute hepatic inflammation states, probably through the TLR4 signaling pathway. Furthermore, EGCG also alleviated hepatic insulin resistance. These data indicate that EGCG improves NAFLD from two ways: inhibition of inflammation and improvement of insulin resistance in liver tissues.

Epigallocatechin-3-gallate (EGCG), a major active ingredient in green tea, has various health benefits. It affects glucose metabolism, but the mechanism is not well understood. This study aimed to identify targets of EGCG related to glucose metabolism. The core fragment of EGCG is a flavonoid. The flavonoid scaffold was used as a substructure to find proteins cocrystallized with flavonoids in the Protein Data Bank. The proteins identified were screened in PubMed for known relationships with diabetes. Dipeptidyl peptidase-4 (DPP4; PDB 5J3J) was identified following this approach. By molecular docking, the interactions of EGCG and DPP4 were assessed. To test the stability of the interactions between EGCG and DPP4, molecular dynamics simulation for 100 ns was performed using Desmond software. In vitro, the concentration of EGCG required to inhibit DPP4 activity by 50% (the IC50 value) was 28.42 μM. These data provide a theoretical basis for intervention in glucose metabolism with EGCG.

Glucose transporters mediate the intracellular uptake of glucose and play important roles in glucose metabolism and signaling. Our understanding of the structural basis for a variety of glucose transporters is, however, still limited. In this study, we presented that the glucose transporter Hgt-1, obtained from the filamentous fungus Neurospora crassa, exhibited much higher glucose transport activity than the other sugar transporter Stp1, obtained from Trichoderma reesei, although they shared high sequence identity of 75.9%. To identify the structural determinants for differential glucose transport capability displayed by Stp1 and Hgt-1, segment swapping was performed by replacing specific sequence regions of Stp1 with the corresponding sequences of Hgt-1. Investigation of the glucose transport capability of the resulting chimeric transporters revealed that replacement of a N- or a C-terminal segment of Stp1, located on the intracellular side, with that of Hgt-1 can apparently improve its transport capability. The combined swapping of these two segments further enhanced the transport capability of Stp1 to a level almost comparable to that of Hgt-1. However, deletion of the C-terminal 32 residues abolished the transport capability of Stp1, whereas deletion of the C-terminal 16 residues apparently improved its transport capability. These results provide insight into the roles of intracellular N- and C-termini of glucose transporters in glucose transport.

Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthesis mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments, and gene manipulation revealed that l-threonine is the starting point involving l-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of l-threonine. The product of this reaction is l-2-amino-acetoacetate, which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate coenzyme A (CoA) ligase (KBL) catalyzes the cleavage of l-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, TMP can also be generated by Bacillus subtilis 168 by using l-threonine and d-glucose as the substrates and TDH as the catalytic enzyme. IMPORTANCE Despite alkylpyrazines' contribution to flavor and their commercial value, the synthesis mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates, and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differ from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthesis mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine’s synthesis pathway involving l-threonine-3-dehydrogenase as the catalytic enzyme and l-threonine as the substrate.

Positron emission tomography (PET) radiopharmaceuticals can noninvasively measure free fatty acid (FFA) uptake into adipose tissue. We studied 29 volunteers to test whether abdominal and femoral subcutaneous adipose tissue FFA uptake measured using [1-11C]palmitate PET agrees with FFA storage rates measured using an intravenous bolus of [1-14C]palmitate and adipose biopsies. The dynamic left ventricular cavity PET images combined with blood sample radioactivity corrected for the 11CO2 content were used to create the blood time activity curve (TAC), and the constant ( Ki) was determined using Patlak analysis of the TACs generated for regions of interest in abdominal subcutaneous fat. These data were used to calculate palmitate uptake rates in abdominal subcutaneous adipose tissue (µmol·kg−1·min−1). Immediately after the dynamic imaging, a static image of the thigh was taken to measure the standardized uptake value (SUV) in thigh adipose tissue, which was scaled to each participant’s abdominal adipose tissue SUV to calculate thigh adipose palmitate uptake rates. Abdominal adipose palmitate uptake using PET [1-11C]palmitate was correlated with, but significantly ( P < 0.001) greater than, FFA storage measured using [1-14C]palmitate and adipose biopsy. Thigh adipose palmitate measured using PET calculation was positively correlated ( R2 = 0.44, P < 0.0001) with and not different from the biopsy approach. The relative differences between PET measured abdominal subcutaneous adipose tissue palmitate uptake and biopsy-measured palmitate storage were positively correlated ( P = 0.03) with abdominal subcutaneous fat. We conclude that abdominal adipose tissue FFA uptake measured using PET does not equate to adipose FFA storage measured using biopsy techniques.

PET radiopharmaceuticals can noninvasively measure free fatty acid (FFA) tissue uptake. Investigators often use PET scan-derived data to calculate FFA flux. We tested whether the [1-11C]palmitate PET measures of palmitate flux provide results equivalent to a continuous infusion of [U-13C]palmitate. Nine volunteers participated in study 1 to evaluate whether a rapidly (10–20 s) given bolus of [1-11C]palmitate affects calculated flux results. Thirty volunteers participated in study 2, which was identical to study 1 except that the [1-11C]palmitate bolus was given over 1 min. Volunteers in both studies also received a continuous intravenous infusion of [U-13C]palmitate. Plasma palmitate concentrations and enrichment were measured by liquid chromatography-mass spectrometry. The PET/CT images were analyzed on a workstation running PMOD. Palmitate flux was estimated using PET time-activity curve (TAC) data from regions of interest in the left ventricle (LV) and aorta both with and without hybrid TACs that employed the11CO2-corrected data for the first 5 min and the11CO2-corrected blood radioactivity for the remainder of the PET scan. Palmitate flux in study 1 measured with PET [1-11C]palmitate and [U-13C]palmitate were not correlated, and the PET [1-11C]palmitate flux was significantly less than the [U-13C]palmitate measured flux. In study 2, the palmitate flux using PET [1-11C]palmitate hybrid LV models provided closer mean estimates of [U-13C]palmitate measured flux. The best PET calculation approaches predicted 64% of the interindividual variance in [U-13C]palmitate measured flux. Palmitate kinetics measured using [1-11C]palmitate/PET do not provide the same palmitate kinetic results as the continuous infusion [U-13C]palmitate approach.

Background Small RNA and degradome sequencing have identified a large number of miRNA-target pairs in plant seeds. However, detailed spatial and temporal studies of miRNA-mediated regulation, which can reflect links between seed development and germination are still lacking. Results In this study, we extended our investigation on miRNAs-involved gene regulation by a combined analysis of seed maturation and germination in barley. Through bioinformatics analysis of small RNA sequencing data, a total of 1324 known miRNA families and 448 novel miRNA candidates were identified. Of those, 16 known miRNAs with 40 target genes, and three novel miRNAs with four target genes were confirmed based on degradome sequencing data. Conserved miRNA families such as miR156, miR168, miR166, miR167, and miR894 were highly expressed in embryos of developing and germinating seeds. A barley-specific miRNA, miR5071, which was predicted to target an OsMLA10-like gene, accumulated at a high level, suggesting its involvement in defence response during these two developmental stages. Based on target prediction and Kyoto Encyclopedia of Genes and Genomes analysis of putative targets, nine highly expressed miRNAs were found to be related to phytohormone signalling and hormone cross-talk. Northern blot and qRT-PCR analysis showed that these miRNAs displayed differential expression patterns during seed development and germination, indicating their different roles in hormone signalling pathways. In addition, we showed that miR393 affected seed development through targeting two genes encoding the auxin receptors TIR1/AFBs in barley, as over-expression of miR393 led to an increased length–width ratio of seeds, whereas target mimic (MIM393)-mediated inhibition of its activity decreased the 1000-grain weight of seeds. Furthermore, the expression of auxin-responsive genes, abscisic acid- and gibberellic acid-related genes was altered in miR393 misexpression lines during germination and early seedling growth. Conclusions Our work indicates that miRNA-target pairs participate in gene expression regulation and hormone interaction in barley embryo and provides evidence that miR393-mediated auxin response regulation affects grain development and influences gibberellic acid and abscisic acid homeostasis during germination. Electronic supplementary material The online version of this article (10.1186/s12870-017-1095-2) contains supplementary material, which is available to authorized users.

Exposure of urothelial carcinoma cells to bacillus Calmette-Guérin affects cellular redox status and tumor cell biology but the mechanism(s) remain unclear. We examined free radical production by bacillus Calmette-Guérin in tumor cells in response to the bacillus using global profiling of reactive oxygen species/reactive nitrogen species. The relationship between free radical generation and downstream cellular events was evaluated.Using fluorescent probes we performed global profiling of reactive oxygen species/reactive nitrogen species in heat killed and viable bacillus Calmette-Guérin, and in the 253J and T24 urothelial carcinoma cell lines after exposure to the bacillus. Inhibition of bacillus Calmette-Guérin internalization and H2O2 pharmacological scavenging were studied for their effect on cellular reactive oxygen species/reactive nitrogen species generation and various physiological end points.Viable bacillus Calmette-Guérin produced H2O2 and O2(-) but nitric oxide was not generated. Loss of viability decreased H2O2 production by 50% compared to viable bacillus. Bacillus Calmette-Guérin internalization was necessary for the bacillus to induce reactive oxygen species/reactive nitrogen species generation in urothelial carcinoma cells. Pharmacological H2O2 scavenging reversed reactive oxygen species/reactive nitrogen species mediated signaling in urothelial carcinoma cells. Bacillus Calmette-Guérin dependent alterations in tumor biology, including intracellular signaling, gene expression and cytotoxicity, depended on free radical generation.This study demonstrates the importance of free radical generation by bacillus Calmette-Guérin and intracellular generation of cellular oxidative stress on the urothelial carcinoma cell response to the bacillus. Manipulating the cellular oxidative stress induced by bacillus Calmette-Guérin represents a potential target to increase the efficacy of the bacillus.

The nano-catalysts of PdxNiy bimetallic nanoparticles (NPs, the nominal atomic ratios of Pd to Ni are 2:1, 3:2 and 1:1) supported on multi-walled carbon nanotubes (MWCNTs) (denoted as PdxNiy/MWCNTs) have been synthesized by a thermal decomposition process using room temperature ionic liquids (RTILs) of N-butylpyridinium tetrafluoroborate (BPyBF4) as the solvent. X-ray diffraction (XRD), scanning electron microscope (SEM) and transmission electron microscopy (TEM) were employed to characterize the morphology of the samples, revealing that the prepared PdxNiy NPs were quite uniformly dispersed on the surface of MWCNTs with an average particle size of ∼8.0 nm. Formic acid oxidation reaction (FAOR) was investigated on the as-prepared catalysts by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), demonstrating that the peak current on the Pd3Ni2/MWCNTs catalyst was about three times higher than that on the Pd/MWCNTs. The lower electrode potential and easier hydrogen evolution, based on the results obtained from chronopotentiometry and CV, respectively, were thought as the main reasons for the excellent electrocatalysis of the Pd3Ni2/MWCNTs toward formic acid oxidation reaction (FAOR) when compared to other samples.

Purpose: The attenuated mycobacterium bacillus Calmette-Guerin is widely used as intravesical immunotherapy for nonmuscle invasive urothelial carcinoma. Previous studies demonstrated that in the laboratory and clinical settings bacillus Calmette-Guerin viability is a variable that correlates with antitumor efficacy. We evaluated how loss of viability impacted a number of molecular and phenotypic intermediate end points that characterize and/or contribute to the direct effect of bacillus Calmette-Guerin on urothelial carcinoma cells.Materials and Methods: We studied the effect of loss of bacillus Calmette-Guerin viability on the tumor cell response to the treatment in 2 human urothelial carcinoma cell lines. The cellular response to bacillus Calmette-Guerin rendered nonviable by heat killing was compared to the response to viable bacillus. Response end points included the induction of oxidative stress, activation of intracellular signaling pathways, gene transactivation and phenotypic changes.Results: Loss...

Filamentous fungus Trichoderma reesei is well known for its high capacity of producing cellulases for industrial application. Besides crystalline cellulose, several soluble sugars including cellobiose can effectively induce cellulase formation. In this study, gluconolactone, previously reported as a β-glucosidase inhibitor, was demonstrated to be capable of inducing cellulase gene expression at a level equivalent to that induced by cellobiose. Gluconolactone-induced formation of cellulase was abolished in T. reesei strain lacking Xyr1 or Crt1, two key regulators for cellulase gene expression. The induced expression of cellulase gene cbh1 was eliminated in the absence of intracellular β-glucosidase Cel1a on gluconolactone while it was hardly affected by the absence of extracellular β-glucosidase Bgl1. We further found that the absence of a cellobiose/glucose transporter Stp1 compromised cellulase production and led to a lower consumption of extracellular gluconolactone. These results suggest that the gluconolactone-derived inducing signal involves both its sensing at the membrane and its intracellular delivery for further processing to initiate cellulase formation.

Scope In this study, we investigated the beneficial effects and the underlying mechanism of epigallocatechin gallate (EGCG) in adipose tissues of rats fed with a high-fat diet (HFD). Methods and results Fasting plasma insulin, epididymal fat coefficient and free fatty acids, homeostasis model assessment-insulin resistance index, and the average glucose infusion rate were determined. EGCG significantly decreased free fatty acids, fasting insulin, homeostasis model assessment-insulin resistance index, and epididymal fat coefficient, and increased glucose infusion rate in HFD group. The levels of toll-like receptor 4, TNF receptor associated factor 6, inhibitor-kappa-B kinase β, p-nuclear factor κB, tumor necrosis factor α, and IL-6 in the EGCG group were all significantly lower than the HFD control group. EGCG also decreased the level of phosphorylated insulin receptor substrate 1 and increased phosphoinositide-3-kinase and glucose transporter isoform 4 in the HFD group. Decreased macrophage infiltration was in EGCG group versus HFD group, and the protein level of CD68 in EGCG group was also significantly lower than that of HFD group. Conclusion EGCG attenuated inflammation by decreasing the content of macrophages, interfered the toll-like receptor 4 mediated inflammatory response pathway, thus, improving insulin signaling in adipose tissues.

(-)-Epigallocatechin gallate (EGCG), the major catechin derived from green tea, reduces the incidence of cardiovascular diseases such as atherosclerosis. Plasminogen activator inhibitor-1 (PAI-1) accelerates thrombus formation upon ruptured atherosclerotic plaques. However, it is not known whether or not EGCG inhibits PAI-1 production induced by tumor necrosis factor-α (TNF-α) in endothelial cells. This study tested the hypothesis that EGCG might have an inhibitory effect on PAI-1 production induced by TNF-α. Human umbilical vein endothelial cells were cultured and incubated with TNF-α and/or EGCG. The expression of p-extracellular regulated protein kinases (p-ERK1/2) and tumor necrosis factor receptor (TNFR1) protein was quantified by Western blotting, and PAI-1 levels were measured by enzyme-linked immunosorbent assay. The results showed that TNF-α increased PAI-1 production in both a dose-dependent and time-dependent manner, and EGCG prevented TNF-α-mediated PAI-1 production and reduced phosphorylation of ERK1/2. The ERK1/2 inhibitor, PD98059 (20 μmol/L), downregulated TNF-α-induced PAI-1 expression 57.69 ± 2.46% (P < 0.01), but had no effect in cells pretreated with EGCG. TNF-α stimulation resulted in a significant decrease in TNFR1, an effect that was abolished by pretreatment with EGCG. These results suggest that EGCG could provide vascular benefits in inflammatory cardiovascular diseases such as decreased thrombus formation associated with ruptured atherosclerotic plaques.

Room temperature ionic liquids (RTILs) of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIBF4) is used as the solvent for the first time to prepare multi-walled carbon nanotubes (MWCNTs) supported nanocomposite catalysts of PdxNiy (atomic ratios of Pd to Ni are 1:1, 1:1.5, 1:2, and 1:2.5) nanoparticles (denoted as PdxNiy/MWCNTs) by using a simple pyrolysis process. The PdxNiy/MWCNTs catalysts are characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Results show that the PdxNiy nanoparticles (NPs) are quite uniformly dispersed on the surface of MWCNTs with an average crystallite size of ∼7.0 nm. The electro-catalytic activity of the PdxNiy/MWCNTs catalysts for ethanol oxidation reaction (EOR) is examined by cyclic voltammetry (CV). It is revealed that the onset potential is ∼80 mV lower and the peak current is about three times higher for ethanol oxidation for MWCNT catalysts with Pd1Ni1.5 compared to those of Pd/MWCNTs. The catalytic mechanisms of the Pd1Ni1.5/MWCNTs towards EOR are also proposed and discussed.

N,W codoped $\mathrm{TiO}_{2}$ nanorods were synthesized via a one-step hydrothermal method using ammonium metatungstate as the nitrogen and tungstate sources. The prepared samples were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), UV–visible diffuse reflectance spectroscopy (DRS), and X-ray photoelectron spectroscopy (XPS). The results indicated that the N,W codoped $\mathrm{TiO}_{2}$ nanorods exhibited a higher photocatalytic activity under visible light irradiation compared with P25 and undoped $\mathrm{TiO}_{2}$ , because the codoping of N and W ions not only extended the visible light absorption but also promoted the separation of the photogenerated electrons and holes.

Multi-walled carbon nanotubes (MWCNTs) decorated with PdxCoy (the nominal atomic ratios of Pd to Co were 3:1, 3:1.5, 3:2, 3:3, respectively) nanoparticles (denoted as PdxCoy/MWCNTs ) were fabricated by a simple pyrolysis process, in which room temperature ionic liquids (RTILs) of butyl-3-methylimidazolium hexafluorophosphate (denoted as [BMIM]PF6) was used as the solvent. X-ray diffraction (XRD) and transmission electron microscopy (TEM) were all used to characterize the PdxCoy/MWCNTs catalysts, showing that the PdxCoy particles were dispersed on the surface of the MWCNTs with an average particle size of ~25.0 nm. The electro-catalytic activity of the PdxCoy/MWCNTs catalysts toward ethanol oxidation reaction (EOR) was examined by cyclic voltammetry (CV). It was revealed that the onset potential was ~90 mV lower and the peak current was about four times higher for ethanol oxidation for Pd3Co1.5/MWCNTs compared to those of Pd3Co1/MWCNTs. The possible catalysis mechanisms of the Pd3Co1.5/MWCNTs toward EOR were also discussed.

Gold (Au) huge particles were prepared by a pyrolysis process of AuCl3 dissolved in distilled water in the presence of multi-walled carbon nanotubes (MWCNTs). X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to characterize the obtained samples. The results showed that Au huge particles with a diameter close to 500 nm were fabricated. The electrocatalytic performance of the huge Au particles modified graphite electrode in the oxygen reduction reaction (ORR) was investigated using cyclic voltammetry (CV). It indicated that the pyrolysis time was a key factor that can affect the electrocatalysis of Au huge particles towards ORR. Developing a novel and simple method of pyrolysis for fabricating Au huge particles is the main contribution of this work, which will be very helpful in the preparation of Au huge particles on a large scale.

Well-defined Au nanoplates have been mass-synthesized through a chemical method at high and low temperatures. A time-saving method for the fabrication of hexagonal Au nanoplates has been developed by modifying polyol process with the presence of binary surfactants. In general methods, most Au nanoplates are in regular shapes of triangle and hexagon. While Au nanoplates with novel shapes, involving star-like and shield-like and other novel polygonal, are found with introduction of obvious temperature variation in the early crystal growth stage. Structural studies demonstrate that the obtained Au nanoplates are single-crystalline with (1 1 1) planes as two basal surfaces. The formation of these new and well-defined Au nanoplates can be understood from the growth of (1 1 1) plane along 〈1 1 0〉, 〈2 1 1〉 and other high-index directions. The ability to control the shape of Au nanoplates provides a great opportunity to systematically investigate the anisotropic growth of noble nanostructures.

High glucose plays an important role in the pathogenesis of atherosclerosis. In this study, we assessed the effects of high glucose on human umbilical vein endothelial cell (HUVEC) apoptosis. Additionally, we investigated whether alpha-lipoic acid, an antioxidant, prevents high glucose-induced apoptosis of HUVECs. HUVECs were treated with high glucose in the presence or absence of alpha-lipoic acid. Treatment of HUVECs with high glucose changed cell morphology and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by high glucose in a dose-and time-dependent fashion. High glucose markedly elevated Bax, and decreased NF-kappaB and Bcl-2 expression. Most importantly, pretreatment with alpha-lipoic acid protected against high glucose-induced apoptosis in the endothelial cells. alpha-Lipoic acid significantly promoted the expression of NF-kappaB while decreasing the expression of Bax and the activities of caspase-3 and-9 without significantly affecting the Bcl-2 level. Our data suggest that high glucose induces apoptosis in endothelial cells. alpha-Lipoic acid effectively attenuates high glucose-induced endothelial cell apoptosis. These findings provide new perspectives on the role of alpha-lipoic acid in cardiovascular disease.

Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by lipopolysaccharide (LPS). An antibody against the 67 kDa laminin receptor (67LR, to which EGCG exclusively binds) was used to examine the effect of EGCG on TLR4 signaling, and a TLR4/MD-2 antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated 3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte inflammation by reducing inflammatory mediator and cytokine levels (IKKβ, p-NF-κB, TNF-α, and IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines, decreased glucose transporter isoform 4 (GLUT4) expression, and inhibited insulin-stimulated glucose uptake. TLR4 inhibition attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels. These data suggest that EGCG suppresses TLR4 signaling in LPS-stimulated adipocytes via 67LR and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression.

In this study, we hypothesized that epigallocatechin gallate (EGCG) would suppress inflammation in the pancreas, and thus, we investigated the effects that EGCG administration had in the pancreas of rats fed a high-fat diet (HFD). To test our hypothesis, 30 male Sprague-Dawley rats were divided into 2 groups: normal diet (control) group and HFD group. When there was a significant difference in body weight between the 2 groups (P < .05), the HFD group was further divided into 2 subgroups: the HFD group (HFD, n = 10, 16 weeks) and the EGCG group (HFD + 3.2 g/kg EGCG, n = 10, 16 weeks). Metabolite levels and the expression of inflammatory markers (tumor necrosis factor alpha [TNF-α], interleukin 6 [IL-6], and toll-like receptor 4) were measured using standard biochemical techniques. Insulin secretion and pancreatic histology were also evaluated. Epigallocatechin gallate significantly decreased fasting insulin levels as well as the homeostasis model assessment-insulin resistance index. In the HFD group, the average glucose infusion rate and the TNF-α and IL-6 levels increased, whereas toll-like receptor 4 and TNF receptor-associated factor-6 did not. A pathologic analysis of pancreatic tissue revealed an increase in inflammatory TNF-α and infiltrating CD68+ macrophages in the islets of the HFD rats, but rarely is this observed in the in the HFD + EGCG rats. Overall, these data suggest that EGCG suppresses inflammation, partially reverses metabolic abnormalities, and ultimately increases insulin sensitivity in the pancreas of HFD rats.

Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

released by a portion of urothelial carcinoma (UC) tumor cells following BCG exposure. Elevated levels of HMGB1 have been observed in the urine of patients following intravesical BCG therapy. Most importantly, in vivo in models have demonstrated that tumor cells engineered to have reduced HMGB1 release fail to respond to intravesical BCG administration. Given the apparent importance of HMGB1 release for in vivo anti-tumor activity, this study evaluated the role of specific elements of BCG/tumor cell interaction in contributing to maximal HMGB1 release. METHODS: Two human UC cell lines, T24 and 253J, were employed. HMGB1 concentrations in cell culture supernatant, with and without BCG treatment, served as the principal end point to assess the role of potentially involved variables. Specific techniques were utilized to determine the role of 5 1 antigen receptor crosslinking, BCG adherence, BCG internalization, BCG viability, iNOS expression/NO production, and p21 expression in mediating HMGB1 release by UC cells in response to BCG. RESULTS: Crosslinking of 5 1 integrin was insufficient to induce HMGB1 release. HMGB1 release by crosslinked cells was not significantly different than untreated controls (p 0.5). Treatment of cells with the peptide GRDGS to block 5 1 integrin and inhibit BCG adherence significantly inhibited HMGB1 release (p 0.001). Inhibition of BCG internalization by pretreatment of cells with Cytochalasin B significantly decreased HMGB1 release relative to BCG alone (p 0.0001). Both viable and heat killed BCG significantly increased HMGB1 release by UC cells relative to controls (p 0.01) The use of heat killed BCG significantly reduced HMGB1 release to 50% of values observed in response to viable BCG (p 0.0001). HMGB1 release by iNOS inhibited cells was significantly reduced (p 0.01). Inducible knockdown of p21 expression in response to BCG significantly reduced HMGB1 release following BCG treatment (p 0.01). CONCLUSIONS: BCG induced non-apoptotic cell death and HMGB1 release occurs as a consequence of a complex multi-step process. Optimal HMGB1 release requires the adherence and internalization of viable BCG, iNOS activity, and p21 expression. An understanding of the steps and mechanisms involved in BCG induced HMGB1 release affords an opportunity for targeted strategies to improve BCG treatment efficacy.

Our group previously noted that HMGB1 release by urothelial carcinoma cells occurs as a consequence of bacillus Calmette-Guérin induced nonapoptotic cell death. Additional studies demonstrated that HMGB1 release in response to bacillus Calmette-Guérin is required for the in vivo tumor response to bacillus Calmette-Guérin. We evaluated the steps required for HMGB1 release by human urothelial carcinoma cells in response to bacillus Calmette-Guérin exposure.We used the T24 and 253J human urothelial carcinoma cell lines. HMGB1 concentrations in cell culture supernatant with and without bacillus Calmette-Guérin treatment served as the principal end point to assess the role of potentially involved variables. Specific techniques were used to determine the role of α5β1 antigen receptor cross-linking, TLR signaling, inducible nitric oxide synthase expression/nitric oxide production, p21 expression, and bacillus Calmette-Guérin adherence, internalization and viability.Cross-linking of α5β1 integrin or signaling through TLR2/4 did not contribute to HMGB1 release. Optimal HMGB1 release required bacillus Calmette-Guérin adherence and internalization. Bacillus Calmette-Guérin viability correlated with the magnitude of HMGB1 release. Inhibition of oxidative stress and p21 expression in response to bacillus Calmette-Guérin decreased the magnitude of HMGB1 release.Bacillus Calmette-Guérin induced nonapoptotic cell death and HMGB1 release occur as a consequence of a complex multistep process. Understanding the steps and mechanisms involved in the induced HMGB1 release would provide an opportunity for targeted strategies to improve bacillus Calmette-Guérin treatment efficacy.

Objectives Evidence suggests that oxidative stress occurring as a consequence of inducible nitric oxide synthase/nitric oxide (iNOS/NO) contributes to the biologic effects of bacille Calmette-Guerin (BCG). Objective of this study is to examine iNOS expression, NO production, and the biologic effect of NO on established intermediate end points for the human urothelial carcinoma cell response to BCG. Materials and methods Quantitative reverse transcriptase-polymerase chain reaction and real-time measurement of NO was used to assess iNOS and NO production, respectively, in 2 human urothelial carcinoma (UC) cell lines, in response to BCG. The effect of blocking NO production using the specific iNOS inhibitor 1400W was determined for multiple intermediate end points characterizing BCG’s direct effects on tumor cell biology. Activation of nuclear factor kappa B and nuclear factor (erythroid-derived 2)–like 2 signaling pathways, transactivation of genes, including p21, CD54, IL6, IL8, CXCL1, CXCL3, CCL20, and cytotoxicity, as measured by vital dye exclusion, lactate dehydrogenase release, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were measured in response to BCG with and without iNOS inhibition. Results Exposure of UC cells to BCG significantly increased both iNOS expression and NO production. Inhibition of iNOS activity with 1400W significantly inhibited BCG’s direct biologic effect on UC cells for all of the end points evaluated. Conclusions iNOS expression, NO production, and the associated oxidative stress play a central role in the response of UC cells to BCG exposure. Manipulation of oxidative stress may afford an opportunity to enhance the antitumor effects of BCG.

Lithium titanate (Li4Ti5O12) was prepared by a quasi solid-state method using water and ethanol as the solvents, in which Li2CO3 and TiO2 were used as the starting materials. In this work, the calcination temperature, molar ratio of Li to Ti, and sintering time were all well investigated. The obtained samples were thoroughly characterized by XRD and SEM, revealing that the above three factors not only affected the crystal structure, but also the morphologies of the resultant samples. Galvanostatic charge discharge curves were also employed to evaluate the electrochemical performance of the samples. The best electrochemical performance of the samples was observed when the molar ratio of Li to Ti, sintering temperature and time are 6:5, 850 oC and 12 hours, respectively. It was revealed that the smaller particle size and the higher crystallinity of the resultant samples were favorable to enhance the electrochemical performance.