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Metabolite lactic acid has always been regarded as a metabolic by-product rather than a bioactive molecule. Recently, this view has changed since it was discovered that lactic acid can be used as a signal molecule and has novel signal transduction functions both intracellular and extracellular, which can regulate key functions in the immune system. In recent years, more and more evidence has shown that lactic acid is closely related to the metabolism and polarization of macrophages. During inflammation, lactic acid is a regulator of macrophage metabolism, and it can prevent excessive inflammatory responses; In malignant tumors, lactic acid produced by tumor tissues promotes the polarization of tumor-associated macrophages, which in turn promotes tumor progression. In this review, we examined the relationship between lactic acid and macrophage metabolism. We further discussed how lactic acid plays a role in maintaining the homeostasis of macrophages, as well as the biology of macrophage polarization and the M1/M2 imbalance in human diseases. Potential methods to target lactic acid in the treatment of inflammation and cancer will also be discussed so as to provide new strategies for the treatment of diseases.

Catalytic conversion of hydrogen peroxide (H2O2) to more toxic hydroxyl radicals (•OH) is a good choice for sterilization and anti-infection, but endogenous H2O2 is insufficient to achieve satisfactory sterilization efficacy. Despite great efforts, designing and developing antimicrobial materials that specifically and effectively self-supply H2O2 at the wound site remain as tremendous challenges. Here, we report a pH-responsive copper peroxide-loaded wound dressing made from copper hydroxide and gelatin sponge and then reacted with H2O2. In vitro experiments show that the prepared wound dressing has good bactericidal properties against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). Moreover, the as-prepared wound dressing can release •OH specifically in the bacterial-infected skin wound, rather than in normal tissues, and in vivo skin wound-healing experiments proved that the synthesized copper peroxide-loaded gelatin sponge could combat E. coli effectively; in addition, Cu2+ released from the gelatin sponge could stimulate angiogenesis and collagen deposition simultaneously. The study provides a strategy to improve antibacterial efficacy and reduce the toxic side effects through the release of •OH by bacterial self-activation.

Paclitaxel (PTX)-coated drug eluting balloon catheters (DEBc) used in the management of neointimal hyperplasia (NIH) have been associated with safety concerns. Alternative coating agents and targeted delivery systems may improve safety and DEBc efficacy. Utilizing a multi-platform approach we designed, developed and evaluated Lumi-Solve, a novel DEBc, coated with ultraviolet (UV) 365 nm-activated caged metacept-3 (c-MCT-3), an epigenetic agent from the histone deacetylase inhibitor (HDACi) class. In vitro catheter and contrast media transmission of UV365nm was evaluated spectroscopically. UV365nm conversion of c-MCT-3 to MCT-3 was evaluated chromatographically. Cellular toxicity and HDACi activity of c-MCT-3 ∓UV365nm was evaluated in vitro. In vivo UV365nm conversion of c-MCT-3 to MCT-3 was evaluated in an ovine carotid artery model. Catheter material and dilute contrast media did not attenuate UV365nm transmission or c-MCT-3 activation. c-MCT-3 demonstrated less cellular toxicity than MCT-3 and PTX. UV365nm-activated c-MCT-3 demonstrated HDACi activity. In vivo activation of c-MCT-3 produced MCT-3. Lumi-Solve, a novel DEBc device developed utilizing a combination of chemical, fibre-optic and catheter based technology platforms, demonstrated potential for targeted delivery of bioactive HDACi to the blood vessel wall supporting direct application to the management of NIH and warranting additional in vivo studies.

Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.

In the present study, the anti-inflammatory effect of Lycium barbarum polysaccharide (LBP) and the possible molecular mechanism thereof were examined, so as to perceive the pharmacological action of LBP. With acute peritonitis in mice as the inflammatory model, the protective effect of LBP on peritonitis mice was evaluated by recording the effect of behavioral scores, studying the pathological damage of intestine and liver, and detecting the levels of inflammatory cytokines. Additionally, by establishing an lipopolysaccharide (LPS)-induced RAW264.7 macrophage model, the effect of LBP on RAW264.7 cell phenotype and culture supernatant inflammatory markers was observed. Finally, the activation of inflammation-related target genes, such as iNOS, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB) p65, and IκBα, were further detected. The results reveal that pretreatment with LBP could decrease the behavioral score of inflammatory mice, inhibit the secretion of pro-inflammatory factors, and reduce liver and intestine injury. LBP can regulate the effect of lipopolysaccharide on the polarization of RAW264.7 cells, and reduce the production of NO and cytokines (TNF-α, IL-1β, IL-6). Further, LBP pretreatment was found to be able to significantly reduce the expression of iNOS, TLR4, NF-κB p65, and IκBα in macrophages. The present research provides evidence that LBP exerts potential anti-inflammatory activity in LPS-induced RAW264.7 macrophages via inhibiting TLR4 and NF-κB inflammatory sites and improving the behavior score of peritonitis mice. PRACTICAL APPLICATIONS: In recent years, the number of deaths worldwide has continued to rise as a result of inflammation. Despite said rise in deaths, many synthetic drugs with anti-inflammatory properties are significantly expensive and also have a host of side effects. Thus, the development of new anti-inflammatory drugs derived from medicinal plants has broad application potential. As such, in the present study, lipopolysaccharide (LPS)-induced macrophages were used to establish inflammatory cell models to verify the anti-inflammatory effect of Lycium barbarum polysaccharides (LBP). Findings were made that LBP could reduce the expression levels of inflammatory cytokines and NO by regulating macrophage polarization and NF-κB translocation, and thus, could exert anti-inflammatory activity. In addition, by intraperitoneal injection of LPS to establish peritonitis mice models, LBP pretreatment was found to have significantly modified the behavioral score of mice, while decreasing the secretion of inflammatory factors and the damage to several organs. The present study provides a basis for further understanding the effects of LBP in acute inflammation.

Identifying the factors controlling the spatial variability of soil metal elements could be a challenge task due to the interaction of environmental attributes and human activities. This study aimed to investigate the critical explanatory variables controlling total Ca, Cd, Cr, Cu, Zn, Fe, Mn, Mg, Pb, and Zn variations in the arable topsoil using classical statistics, principal component analysis, and random forest techniques. The work was conducted in the core region of the Three Gorges Reservoir of China. The explanatory variables included soil, topography, climate, vegetation, land use type, and distance-related parameters. Average concentrations of the metal elements were in order of Fe > Mg > Ca > Mn > Zn > Cr > Ni > Pb > Cu > Cd. Soil Cr, Fe, and Pb showed low variability while others presented medium variability. Average concentrations of Cr, Fe, Cd, and Mg exceeded their corresponding background values. There were highly positive correlations between all metal elements except Pb, Cd and Cr. The principal component analysis further demonstrated that the sources of Pb, Cd, and Cr differed with other elements. The results of random forest suggested that soil properties followed by topography were critical parameters affecting the variations of Ca, Mg, Mn, Fe, Ni, Zn, and Cu. Agricultural activities and soil properties were major factors controlling the variations of Pb, Cr, and Cd. Further study should be conducted to understand the relations between the metal elements and soil properties.

Background4-vinylcyclohexene diepoxide (VCD) has long been considered a hazardous occupational chemical that promotes ovarian failure. However, VCD is also used as a research compound to chemically induce animal models of premature ovarian insufficiency (POI), and in related work we unexpectedly found that VCD apparently exhibits both dose- and duration-dependent opposing, hormone-like effects on the maintenance of the primordial follicle pool, follicle development, and ovulation induction.ResultsWe conducted experiments with cultured murine ovaries and performed transplantation experiments using postnatal day (PD) 2 and PD12 mice and found that low-dose, short-term exposure to VCD (VCDlow) actually protects the primordial/primary follicle pool and improves the functional ovarian reserve (FOR) by disrupting follicular atresia. VCDlow inhibits follicular apoptosis and regulates the Pten-PI3K-Foxo3a pathway. Short-term VCD exposure in vivo (80 mg/kg, 5 days) significantly increases the number of superovulated metaphase II oocytes, preovulatory follicles, and corpus luteum in middle-aged mice with diminished ovarian reserve (DOR). We demonstrate that low-dose but not high-dose VCD promotes aromatase levels in granulosa cells (GCs), thereby enhancing the levels of estradiol secretion.ConclusionOur study illustrates a previously unappreciated, hormone-like action for the occupational “ovotoxin” molecule VCD and strongly suggests that VCDlow should be explored for its potential utility for treating human ovarian follicular development disorders, including subfertility in perimenopausal women.

A polyhedral Zn-based MOF [Zn (B-BDC) (Dpy)] •H2O (B-BDC = 3, 5-Dicarboxyphenylboronic acid, Dpy = 4, 4′-Bipyridine) (CCDC: 1559247), named Zn-BDC, has been initially designed and constructed via hydrothermal method. Then, its crystal structure, morphology and thermal properties were confirmed by using a series of characterization methods. Furthermore, composite materials, denoted as Zn-BDC-T (T = pyrolysis temperature), were synthesized by directly calcining Zn-BDC under N2 atmosphere at different temperatures. Herein, Zn-BDC-800 was recognized as N-doped Zn3B2O6 composite according to characterization results. When the Zn-BDC-800 was used as electrode materials for sodium-ion batteries, it exhibited a maximum discharge specific capacity of 295.6 mAhg−1 at the current density of 50 mAg−1 and its coulombic efficiency still remained at 99% after 100 cycles. Thus, this N-doped Zn3B2O6 polyhedral composite derived from Zn-MOF has broad development prospects in the field of SIBs.

The carbon-carbon double bond functionalized 24-nuclear copper cluster was successfully synthesized. The cluster can capture an impressive quantity of dyes, such as RhB (rhodamineB), MB (methylene blue) and CV (crystal violet). By the interaction between double bond and ions, the complex can selective separate cationic dyes from aqueous solution effectively.

Soil pH is a key factor that controls soil nutrient availability, soil microbial activities, and crop growth and development. However, studies on the soil pH variations of cultivated lands in different horizons at the regional scale remain limited. In this work, 348 soil samples were collected from three soil horizons (A, B, and C) at 120 sites over the hilly region of Chongqing, southwestern China. Six topographic indicators, four climate parameters, and parent material were considered. Classification and regression trees (CARTs) were applied to investigate the relationships between soil pH and the variables in the A, B, and C horizons. Model performances were evaluated by root mean square error (RMSE), relative root mean square error (RRMSE), and coefficient of determination (R2). Results showed that soil pH increased obviously from the A to C horizons. Soil pH was predicted well by the forcing factors with the CART models in all horizons. RMSE, RRMSE, and R2 varied between 0.37 and 0.435, between 5.93 and 7.23%, and between 0.71 and 0.80, respectively. The relative importance of the studied variables to soil pH differed with the horizons. Annual temperature range (ATR), terrain wetness index (TWI), and Melton ruggedness number were critical factors that controlled soil pH variability in the A horizon. Parent material, precipitation of warmest quarter (PWQ), ATR, and TWI were important variables in the B horizon. Parent material, PWQ, ATR, and precipitation were key factors in the C horizon. The results are expected to provide valuable information for designing appropriate measurements for agricultural practices and preventing soil acidification.

Phytate-mineralizing rhizobacteria (PMR) play an important role in providing phosphorus for the sustainable plant growth. It is important to investigate the ability of PMR to produce phytase under different phosphate levels for its application. The effects of different concentrations of soluble phosphate on the ability of phytate mineralization of Pseudomonas fluorescens JZ-DZ1, a phytate-mineralizing rhizobacterium, were investigated in both solid and liquid media. The results on solid media showed that halo zone width gradually reduced with concentrations of soluble phosphate increasing from 0.05 to 20 mM, indicating the reduction of the ability of phytate mineralization. The results were consistent with the quantitative detection of phytase activity from the overall trend. An 1866-bp β-propeller phytase (BPP) gene (phyPf) was cloned from the strain, and the deduced amino acid sequence of phyPf shared 98 % of identity with a known BPP from Pseudomonas sp. BS10-3 (AJF36073.1). The results of relative real-time quantitative PCR assay showed that the expression of phyPf was induced by a low concentration (0.1 mM) of soluble phosphate, suggesting that BPP secretion was regulated by gene phyPf. The BPP-harboring bacterium P. fluorescens JZ-DZ1 with low phosphate-inducible ability of phytate mineralization could be potentially applied to promote phosphorus uptake for plants in the future.

Hydroxamic acid-based histone deacetylase inhibitors (HDACi) are a class of epigenetic agents with potentially broad therapeutic application to several disease states including post angioplasty mediated neointimal hyperplasia (NIH). Precise spatiotemporal control over the release of HDACi at the target blood vessel site is required for the safe and successful therapeutic use of HDACi in the setting of drug eluting balloon catheter (DEBc) angioplasty treatment of NIH. We aimed to develop and characterise a novel photoactive HDACi, as a potential coating agent for DEBc. Metacept-3 1 was caged with a photo-labile protecting group (PPG) to synthesise a novel UV365nm active HDACi, caged metacept-3 15. Conversion of caged metacept-3 15 to active/native metacept-3 1 by UV365nm was achievable in significant quantities and at UV365nm power levels in the milliwatt (mW) range. In vitro evaluation of the biological activity of pre and post UV365nm activation of caged metacept-3 15 identified significant HDACi activity in samples exposed to short duration, mW range UV365nm. Toxicity studies performed in human umbilical vein endothelial cells (HUVEC's) identified significantly reduced toxicity of caged metacept-3 15 pre UV365nm exposure compared with native metacept-3 1 and paclitaxel (PTX). Taken together these findings identify a novel photo-activated HDACi, caged metacept-3 15, with pharmacokinetic activation characteristics and biological properties which may make it suitable for evaluation as a novel coating for targeted DEBc angioplasty interventions.

Knowledge about the relative importance of influencing-factors on rice yield gap is crucial to rice production, especially in southwestern China where topography is extremely complicated. In the current study, the data of rice yield from a total of 76 experiments were collected in 2008 and 2009 in Chongqing, southwest China. For each location, two treatments with fertilizer and without fertilizer were carried out, each treatment was performed with three replications, and yield gap was calculated using fertilized yield minus unfertilized yield. Seventeen influencing-factors including variety, fertilization, climate, terrain, and soil properties were obtained at each location. Regression tree (RT) model were employed to investigate relative important of influencing-factors to rice yield gap variability. The result of Pearson correlation analysis suggested yield gap of rice was positively correlated with sunshine hours, phosphorous and potassium fertilizers, while negatively correlated with soil available nitrogen content. The results of RT showed that the selected influencing-factors explained about 74.1% of rice yield gap variation. Meanwhile, the result also indicated variety followed by others had more influence on rice yield gap variation. Our findings analyzed by regression model at a regional scale suggested that more precise fertilization recommendation should be formulated based on comprehensive factors (e.g., soil, climate, terrain, variety), which reasonably guided farmer and government for rice production.

Sodium glucose transporter type 2 inhibitors may reduce cardiovascular events in type 2 diabetes. Our study aimed to determine the effect of the sodium glucose transporter type 2 inhibitor dapagliflozin on endothelial cell activation, vasoreactivity and atherogenesis using in vitro and in vivo models and identify associated molecular mechanisms.In vitro studies utilised human vascular endothelial cells stimulated with tumour necrosis factor α or hyperglycaemic conditions. In vivo studies were performed in C57Bl/6J mice to evaluate direct vasorelaxation responses evoked by acute dapagliflozin administration and acute vaso-protective effects of dapagliflozin on hyperglycaemia-induced endothelial dysfunction. Adult and aged Apolipoprotein E-deficient mice maintained on a high-fat diet were used to investigate endothelial-dependent vascular reactivity and atherogenesis. Dapagliflozin treatment (1.0 mg/kg/day) was administered for 4 weeks.In vitro studies demonstrated dapagliflozin-mediated attenuation of tumour necrosis factor α- and hyperglycaemia-induced increases in intercellular adhesion molecule-1, vascular cell adhesion molecule-1, plasminogen activator inhibitor type 1 and NFκB expression. Acute dapagliflozin administration dose-dependently induced endothelium-independent vasorelaxation. Chronic dapagliflozin treatment improved endothelial function and significantly reduced in vivo vascular adhesion molecule and phospho-IκB expression together with macrophage vessel wall infiltration.These observations identify a potential role for dapagliflozin in the attenuation of atherogenesis and identify anti-inflammatory molecular mechanisms associated with these effects.

OBJECTIVES The aim of this study was to investigate the role of P2X7R (purinergic 2X7 receptor) and NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome activation in the process of pancreatic fibrosis in a mouse model of chronic pancreatitis (CP). METHODS Chronic pancreatitis was induced by repeated intraperitoneal injections of 50 μg/kg cerulein for 6 weeks in mice. P2X7R antagonist oxidized ATP (OxATP) or brilliant blue G (BBG) was administered after the last cerulein injection for 2 weeks. Pancreatic chronic inflammation and fibrosis were evaluated by histological score, Sirius red staining, and alpha-smooth muscle actin immunohistochemical staining. We further determined pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, interleukin 1β (IL-1β), and IL-18. RESULTS The pancreatic P2X7R, NLRP3, and caspase-1 expressions in gene and protein levels and the pancreatic concentrations of caspase-1, IL-1β, and IL-18 were all reduced significantly in both the OxATP and BBG groups (P < 0.05). The pancreatic chronic inflammation and the fibrosis indices were all remarkably attenuated (P < 0.05). CONCLUSIONS P2X7R antagonist OxATP and BBG significantly decreased pancreatic chronic inflammation and fibrosis in a mouse CP model and suggested that blockade of P2X7R-NLRP3 inflammasome signaling pathway may represent a novel therapeutic strategy for CP and its fibrotic process.

Apatinib mesylate, a small-molecule tyrosine kinase inhibitor (TKI) of vascular endothelial growth factor receptor-2 (VEGFR-2), has been recommended as a third-line class A agent for patients with advanced gastric adenocarcinoma in China since April 2018. Thus far, there is no case report on the combined use of apatinib and S-1 as the first-line treatment option for metastatic gastric cancer (GC) patients based on peripheral circulating tumor cell (pCTC) monitoring. We present a unique case in which liver metastases originating from GC gradually shrank and disappeared in the presence of pCTC-positive disease after treatment with apatinib for 17 months. More surprisingly, the pCTCs became negative after the addition of S-1 halfway through treatment. A 71-year-old Chinese man who underwent gastroscopic biopsy and laparoscopy was diagnosed with gastric adenocarcinoma with multiple liver metastases. The pathological stage was T4NxM1. The patient obtained clinical benefit from first-line apatinib within 4 weeks. After developing grade 2 hand-foot syndrome, the patient began to take S-1 alone. When the condition was significantly relieved, the elderly patient continued to take apatinib combined with S-1 and achieved satisfactory therapeutic results. Many studies suggest that the prognosis of this type of GC is grim. However, apatinib increased the overall survival (OS) duration of the patient to 34 months. We hypothesize that combining apatinib with S-1 as a first-line therapeutic option for pCTC-positive advanced GC is feasible.

Elevated serum levels of vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2) are associated with poor prognosis in patients with gastric cancer. Little is known regarding the clinical benefits of combining celecoxib, a selective inhibitor of COX-2, with standard chemotherapy regimens for the treatment of gastric cancer patients. In this study, we investigated the effect of the combinatorial use of celecoxib with standard chemotherapy on the serum levels of VEGF and COX-2 in patients with gastric cancer. In our study, 80 patients with gastric cancer who underwent laparoscopic radical surgery were randomized into two groups, the combination [celecoxib plus standard oxaliplatin, leucovorin and 5-fluorouracil (FOLFOX4) chemotherapy, n=40] and the FOLFOX4 alone (n=40) groups. In the combination group, celecoxib was orally administered to the patients (400 mg, twice daily). The serum levels of VEGF and COX-2 were measured by ELISA prior to and following surgery. We detected no significant difference in the serum levels of VEGF and COX-2 between the combination and FOLFOX4 alone groups prior to chemotherapy (P>0.05). However, after 6 cycles of chemotherapy, there was a greater decrease in the serum levels of VEGF and COX-2 in the combination group compared to those in the FOLFOX4 group (P

Femtosecond laser micromachining technology shows abroad application background in the field of micro manufacturing due to its unique advantages, especially for micromachining of ultrahard materials such as Silicon carbide (SiC) ceramic. The femtosecond laser micromachining system was set up, by using the system, effects of scanning velocity and laser pulse energy on quality of micromachined features were evaluated. The optimized technological parameter was obtained as 8mW, 1mm/s with 1kHz repetition frequency respectively on the basis of the morphological characteristics and microstructure accuracy. Besides, V-shaped cavity of 300μm depth and 120°angle was generated with layer-by-layer scan machining. Thus femtosecond laser micromachining technology is an effective method for hard and brittle materials precision processing.

Intimal hyperplasia appears to differ after implanting a drug-eluting stent (DES) with a biodegradable or a permanent polymer. The aim of the present study was to compare biodegradable with permanent polymer DES, since the available data are limited. One hundred patients with de novo coronary artery stenosis were included in this study. The patients were classified into 2 groups: DES with a biodegradable polymer (n=50) and DES with a permanent polymer (n=50). Optical coherence tomography (OCT) examination was performed before and after stent implantation. A follow‑up OCT, performed 1 year after stent implantation, compared the morphologies of intimal hyperplasia in the 2 groups. The frequencies of uncovered stent struts (2.27 vs. 1.87%, P=0.145) and stent strut malapposition (1.9 vs. 2.02%, P=0.655) upon the first-year follow-up were not significantly different. Average neointimal thickness was lower in the biodegradable compared with the permanent polymer group (106.12±80.65 vs. 181.20±146.96 µm, P

Epigenetic regulation of microRNA (miRNA) expression has recently been implicated in the pathogenesis of myelodysplastic syndrome (MDS). Particular interest has focused on miRNA-124 expression, which is inhibited in MDS and has recently been demonstrated to be upregulated in response to epigenetic treatment (EGT). Previous studies have determined the in vitro and in vivo expression of miRNA-124 and several molecular targets, including cyclin-dependent kinase (CDK) 4, CDK6 and enhancer of zeste homolog 2 (EZH2), in order to elucidate the molecular mechanisms associated with the miRNA-124-mediated therapeutic response to EGT in MDS and identify additional potential biomarkers of early EGT treatment response in myeloid malignancies. In vitro studies in the HL60 leukemic cell line identified upregulation of miRNA-124 expression in response to single-agent EGT with either azacytidine (AZA) or the histone deacetylase inhibitor panobinostat (LBH589). Combination EGT with AZA and LBH589 resulted in significant additive induction of miRNA-124 expression. Expression of downstream targets of miRNA-124, including CDK4, CDK6 and EZH2, in response to single agent and combined EGT was determined in HL60 cells. Single and combination EGT treatment resulted in inhibition of CDK4, CDK6 and EZH2 expression with combination EGT resulting in a significant and additive inhibitory effect. In vivo studies using peripheral blood mononuclear cells from patients receiving combination EGT for high risk MDS or acute myeloid leukemia demonstrated significant induction of miRNA-124 and inhibition CDK4 and CDK6 messenger (m)RNA expression in patients that responded to combination EGT. A trend to inhibited EZH2 mRNA expression was also identified in response to combination EGT. Overall, the present observations identify a potential molecular mechanism for miRNA-124-mediated response to EGT involving regulation of CDK4, CDK6 and EZH2 expression. In addition, the present findings further qualify miRNA-124 as a possible biomarker of early response to EGT in myeloid malignancies and potentially a valid therapeutic target, together with CDK4, CDK6 and EZH2.

To improve the friction and wear behavior of hydrogenated nitrile butadiene rubber (HNBR) composites, graphite and silane-treated Silicon carbon (SiC) were incorporated. The tribological properties of the resulting composites were investigated systematically on a model ball-on-block test rig. The friction and wear mechanisms of the composites were studied through analyzing the worn surfaces by a scanning electron microscopy. Experimental results showed that the friction and wear behavior of the filled composites were improved greatest when graphite and silane-treated SiC were added together, indicating that there was a synergistic effect between them.

A series of novel N-acyl substituted quinolin-2(1H)-one derivatives were synthesized and screened in vitro for their antibacterial and antifungal activities by disc diffusion method. All the compounds exhibited moderate to good antimicrobial activities, some of these compounds displayed comparable or better antibacterial or antifungal activities against some tested strains compared to the reference drugs Streptomycin and Fluconazole.

Knowledge of arsenic and heavy metal concentrations and distributions is essential in the design of environmental regulatory strategies. The present study was undertaken as a preliminary survey on soil contamination in a representative agricultural area of southwestern China. The objectives were: (1) to characterize the variability of As, Cr, Hg, and Pb; (2) to assess the effect of land use (vegetable land and paddy fields) on these elements; and (3) to identify their natural or artificial origins. Samples from 119 sites were collected and analyzed in 2006. Land use map units were delineated at a scale of 1:2000 from field survey. Analysis of variance, correlation analysis, factor analysis, and a geographic information system were adopted to investigate the spatial distributions and sources of the elements. The average concentrations of the elements were 6.26 mg As kg−1, 52.90 mg Cr kg−1, 0.066 mg Hg kg−1, and 33.13 mg Pb kg−1. Mean contents of Hg and Pb were markedly higher than their background levels i...

Cinnamaldehyde (1) is a pharmacologically active ingredient isolated from cassia twig (Ramulus Cinnamomi), which is commonly used in herbal remedies to treat fever-related diseases. Both TRPV1 and TRPM8 ion channel proteins are abundantly expressed in sensory neurons, and are assumed to act as a thermosensor, with the former mediating the feeling of warmth and the latter the feeling of cold in the body. Both of them have recently been reported to be involved in thermoregulation. The purpose of this paper is to further uncover the antipyretic mechanisms of 1 by investigating its effects on the mRNA expression levels and functions of both TRPV1 and TRPM8. The results showed that 1 could up-regulate the mRNA expression levels of TRPV1 at both 37 and 39 degrees C, and its calcium-mediating function was significantly increased at 39 degrees C, all of which could not be blocked by pretreatment of the neuronal cells with ruthenium red, a general transient receptor potential (TRP) blocker, indicating that the action of 1 was achieved through a non-TRPA1 channel pathway. In conclusion, the findings in our in vitro studies might account for part of the peripheral molecular mechanisms for the antipyretic action of 1.

Glucagon-like peptide-1 (GLP-1) administration attenuates endothelial cell dysfunction in diabetic patients and inhibits tumour necrosis factor α (TNF)-mediated plasminogen activator inhibitor type-1 (PAI-1) induction in human vascular endothelial cells. The short half-life of GLP-1 mediated via degradation by the enzyme dipeptidyl peptidase 4 mandates the clinical use of long-acting GLP-1 analogues. The effects of a long-acting GLP-1 analogue on PAI-1 and vascular adhesion molecule expression in vascular endothelial cells are unknown. In this report, we demonstrate for the first time that the treatment with liraglutide, a long-acting GLP-1 analogue, inhibited TNF or hyperglycaemia-mediated induction of PAI-1, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA and protein expression in a human vascular endothelial cell line. In addition, treatment attenuated TNF- or hyperglycaemia-mediated induction of the orphan nuclear receptor Nur77 mRNA expression. Taken together, these observations indicate that liraglutide inhibits TNF- or glucose-mediated induction of PAI-1 and vascular adhesion molecule expression, and this effect may involve the modulation of NUR77. These effects suggest that liraglutide may potentially improve the endothelial cell dysfunction associated with premature atherosclerosis identified in type 2 diabetic patients.

Prostaglandin E2 (PGE2) works as a common final mediator of the febrile. Guizhi-Tang, one of the most famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions, was previously reported to reduce the production of PGE 2 in rats. 2-Methoxycinnamaldehyde is a principle compound isolated from Guizhi-Tang. The aim of the present study was to investigate the effects of 2-methoxycinnamaldehyde on PGE2 production of rat cerebral endothelial cells (CECs). 2-Methoxycinnamaldehyde dose-dependently inhibited interleukin (IL)-1beta-induced PGE2 production in CECs with IC50 values of 174 microM. IL-1beta stimulation increased the protein, activity and mRNA expression of cyclooxygenase (COX)-2 but not COX-1. 2-Methoxycinnamaldehyde reduced IL-1beta-induced protein and activity of COX-2, but did not influence the COX-2 mRNA expression. Our results show that prostaglandin production in CECs during stimulated conditions is sensitive to inhibition by 2-methoxycinnamaldehyde and suggest that 2-methoxycinnamaldehyde may reduce COX-2 protein level and activity but not COX-2 mRNA.

Combination epigenetic treatment (EGT) utilizing DNA methyl transferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) may be more efficacious than single agent treatment in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). The molecular mechanisms behind the potential clinical efficacy of combination EGT treatment are incompletely understood and the frequently lengthy EGT regimes required to determine clinical response have generated a significant demand for early molecular markers of treatment response. Our study aimed to identify the effect of combination azacitidine (AZA) and panobinostat (LBH589) on expression levels of a panel of genes implicated in the pathogenesis of high-risk MDS or AML in HL-60 cells. We also characterized gene expression profiles in peripheral blood mononuclear (PBMCs) from patients in a recently reported phase Ib/II clinical trial using the combination of AZA and LBH589 and correlated these findings with clinical response to treatment. In vitro analysis demonstrated increased expression of caspase-3, Nor-1, NUR77, p15INK4B and p21WAF1/CIP1 and decreased expression of Bcl‑xL in HL-60 cells treated with combination EGT. Analysis of patient samples prior to treatment demonstrated a significant reduction in NUR77 and p21WAF1/CIP1 expression compared to healthy controls. NUR77 and p21WAF1/CIP1 levels were similar between treatment non‑responders and responders at screening. Early post first cycle treatment (day 25) analysis demonstrated a significant increase in expression of both NUR77, and p21WAF1/CIP1. A significant increase in NUR77, and p21WAF1/CIP1 together with a trend to increase in p15INK4B first cycle expression was observed in treatment responders compared to non-responders. In summary, combination AZA and LBH589 epigenetic treatment is associated with in vitro and in vivo modulation of genes implicated in the pathogenesis of MDS/AML. Early expression of NUR77 and p21WAF1/CIP1 correlated with clinical response to combination EGT suggesting investigation for potential use as molecular markers of early treatment response may be warranted.

An ultrasensitive polyclonal antiserum against ciprofloxacin (CPFX) was developed by introducing cationized bovine serum albumin (BSA) as the carrier. Conjugates used as immunogens were synthesized using CPFX coupled to BSA cationized with ethylenediamine and hexamethylenediamine, namely CPFX-eBSA and CPFX-hBSA. The results showed that the antisera immunized with CPFX-hBSA exhibit an improved sensitivity and specificity compared with those immunized with CPFX-eBSA. Under the conditions of optimal heterologous enzyme-linked immunosorbent assay, the optimal antiserum yielded an IC50 of 0.097 ng mL−1, which proves it to be more sensitive than any CPFX antibody. Cross-reactivities with norfloxacin, enrofloxacin, and ofloxacin were < 3.0 %. No cross-reactivities with penicillin and gentamicin were found. The limit of detection was 0.01 ng mL−1, which is far below the maximum residue level established by the EU regulation, suggesting the great potential of the presented antiserum for quantitative assays of CPFX in samples.

A series of 13 title compounds of type (I) are prepared and tested for their antibacterial and antifungal activities.

Presented in part at the 53rd Annual Meeting of the American Society of Hematology, San Diego, CA, December 10-13, 2011, and the 17th Congress of the European Haematology Association, Amsterdam, June 14-17, 2012. Therapeutic options are limited for elderly patients with acute myeloid leukemia (AML). A phase Ib/II study was undertaken to evaluate the maximum-tolerated dose (MTD) and preliminary efficacy of the pan-histone deacetylase inhibitor panobinostat (LBH589) in combination with azacitidine in patients with AML or high-risk myelodysplastic syndrome (MDS) naïve to intensive chemotherapy. Thirty-nine patients (AML=29, MDS=10) received azacitidine 75 mg/m2 subcutaneously (days 1-5) and oral panobinostat (starting on day 5, thrice weekly for seven doses) in 28-day cycles until toxicity or disease progression. Dose-limiting toxicities during the phase Ib stage were observed in 0/4 patients receiving 10 mg panobinostat, in 1/7 patients (fatigue) receiving 20 mg, in 1/6 patients (fatigue) receiving 30 mg and in 4/5 patients (fatigue, syncope, hyponatremia and somnolence) receiving 40 mg. In phase II, an additional 17 patients received panobinostat at a MTD of 30 mg. The overall response rate (ORR=CR+CRi+PR) in patients with AML was 31% (9/29) and that in patients with MDS was 50% (5/10). After a median follow-up of 13 months, the median overall survival was 8 and 16 months in patients with AML and MDS, respectively. Increased histone H3 and H4 acetylation was a useful early biomarker of clinical response. Combining panobinostat with azacitidine was tolerable and clinically active in high-risk MDS/AML patients, warranting further exploration. Refereed/Peer-reviewed

The objectives of the current study are to determine the factors impacting cultivated land productivity and to illustrate the spatial distribution pattern of cultivated land productivity using GIS. Landform information (slope and topographical position), soil physical and chemical properties (effective soil depth, organic matter, parent materials, texture, pH, total nitrogen, available phosphorus, available potassium, and drainage capacity), and cropping system were selected to assess the cultivated land productivity using Delphi technique. The weight of each index was determined by AHP approach. The results showed that about 60.21% of the study area was classified as third level followed by 28.5% of the total area as forth level, located in northwestern and southern Wanyuan, respectively. The highly productivity cultivated land is mainly distributed in valley with slope of less than 15° in Wanyuan county.

The objective of this paper is to evaluate land suitability for sorghum using geographic information system (GIS). Terrain attributes (slope and aspect) and soil physical and chemical properties (effective soil depth, organic matter, texture, available phosphorus, available potassium, and drainage capacity) were selected using Delphi technique. The weight of each index was determined by AHP approach. The results showed that about 94% of the study area was classified as suitable and highly suitable land for planting sorghum. The highly suitable land is mainly located in the north-western Jiangyang.

Histone deacetylase inhibitors (HDACi) demonstrate considerable in vitro and in vivo activity and clinical efficacy in the treatment of hematological malignancies. Pre-clinical and early phase clinical trials identify therapeutic activity using a combination of HDACi and demethylating agents which may be more efficacious than single agent treatment. Our studies aimed to determine the effects and molecular mechanisms of action of novel hydroxamate (MCT-3) and benzamide [MGCD0103 (MG)] HDACi's in the HL-60 cell line alone and in combination with the demethylating agent 5-aza-cytidine (AZA). MG, MCT-3 and AZA treatment significantly inhibited HL-60 cell growth in vitro with MG being the most potent agent. MG in combination with AZA demonstrated no significant increase in inhibition of cell growth over MG treatment alone whilst MCT-3 in combination with AZA demonstrated increased inhibition of cell growth over either agent alone although no more significant than MG alone. MG alone or MCT-3 in combination with AZA significantly increased p15 and caspase-3 expression. MG and MCT-3 significantly attenuated AZA-induced MMP-9 mRNA expression and proteolytic activity. Interestingly, MCT-3, MG and AZA alone and in combination increased expression of the novel tumour suppressor gene Nur77, important in leukemogenesis, with MG a more potent inducer as a single agent. These observations suggest the enhanced anti-leukemia activity of the combination of AZA and HDACi may only reside with certain HDACi classes and may be in-part explained by regulation of genes associated with cell cycle arrest, apoptosis and tumour suppression.

Emodin is a principle ingredient isolated from rhubarb rhizome, which is commonly used for constipation or pain-related diseases in traditional Chinese medicine (TCM) practice. The transient receptor potential vanilloid 1 ion channel proteins (TRPV1) are abundantly expressed in the peripheral sensory neurons and are assumed to act as a kind of nociceptor involved in the perception of pain and development of hyperalgesia. The aim of this study was to further unravel the analgesic mechanisms of rhubarb through investigating the effects of its main constitutive ingredient emodin on the expression of TRPV1 mRNA as well as on its calcium- mediating functions in vitro. The primary DRG neurons with a high purity and viability were obtained, and the TRPV1 mRNA expression levels were examined by using real-time RT-PCR and the elevated amplitudes of intracellular [ Ca2+ ] i in the DRG neurons evoked by TRPV1 agonist capsaicin were examined by confocal microscopy. The results showed that emodin could significantly down-regulate both the mRNA expression of TRPV1 and the capsaicin-evoked intracellular fluorescent intensity in the DRG neurons under both 37°C and 39°C in vitro. Concomitantly, all of the changes induced by emodin could not be blocked by pretreatment of the primary neurons with capsazepine, an antagonist of TRPV1. In conclusion, we established that the mRNA expression level of TRPV1 and its calcium-mediating function in naive DRG neurons could be down-regulated by emodin through perhaps the non-TRPV1 channel pathways, and this might be the molecular mechanisms for rhubarb to inhibit hyperalgesia induced by inflammatory stimuli.

Total of 120 soil samples from the topsoil (0-20cm) at an approximate interval of 100 m were collected in Wutan, China, to determine the spatial variability of five heavy metals, Cd, As, Hg, Pb, and Cr, with classical statistical and geostatistical analyses. Ordinary kriging was utilized for estimating heavy metals determination. Classical statistical indicated that As, Pb, and Cr concentrations were correlated with organic matter (p≪0.05). The concentration of Pb was negatively correlated with pH (p≪0.05). Geostatistical analyses suggested that the coefficient variation of heavy metals in the study area were relatively small. The Nugget/Still ratio of all heavy metals were less than 75% except Cd, which indicated that the spatial variability of the heavy metals were influenced by both structure factors and human activities. Higher heavy metal concentrations were mainly located in paddy fields and residential areas. To some degree, human activities, such as fertilizer application, sewage irrigation, and vehicles, influenced the spatial distributions of the analyzed heavy metals.

There is a growing concern about environmental contamination in the three gorges area. The objectives of this study were to investigate the spatial variability and the possible influence factors of seven heavy metals (As, Cd, Cr, Cu, Hg, Pb, and Zn) in the center of this area based on multivariate and geostatistical approaches. All analyzed heavy metals were below their background levels, except Cd. The average concentrations of the analyzed elements in topsoil (0-20 cm) were 5.83 mg As kg(-1), 0.21 mg Cd kg(-1), 78.79 mg Cr kg(-1), 21.53 mg Cu kg (-1), 0.049 mg Hg kg(-1), 24.12 mg Pb kg(-1), and 68.5 mg Zn kg(-1). The concentration of As was mostly due to parent materials, whereas the source of Pb was mainly due to vehicle exhaust. The high concentration of Cd was resulted from agricultural practices and parent materials. The concentrations of Cr, Cu, Hg, and Zn were associated with parent materials and human activities.

To investigate the effects of sphingosine-1-phosphate (S1P) and its analogue FTY720 on the lung injury induced by acute necrotizing pancreatitis in rats.Acute necrotizing pancreatitis was induced by retrogradely injection of 5% sodium taurocholate of biliopancreatic duct in rats. Sphingosine-1-phosphate (100 microg/kg) or FTY720 (1 mg/kg) was administered immediately after the model induction by peritoneal injection. Six hours after the model induction, bronchoalveolar lavage protein concentration, total cell count, polymorphonuclear neutrophil percentage, proinflammatory cytokines (interleukin 1beta, interleukin 6, and tumor necrosis factor alpha), nuclear factor kappaB activation of alveolar macrophages, and lung myeloperoxidase (MPO) activity were examined. Serum amylase and lipase were tested. In addition, histopathological changes of the pancreas and lung were observed.Bronchoalveolar lavage protein concentration, total cell count, PMN percentage, proinflammatory cytokines, nuclear factor kappaB activation, lung capillary leakage, and lung myeloperoxidase were all reduced significantly in both S1P and FTY720 groups. The pulmonary pathological injury in both S1P and FTY720 groups was ameliorated obviously. Nevertheless, the serum amylase, lipase, and the pancreatic pathological damages were not decreased.Sphingosine-1-phosphate and its analogue FTY720 significantly decreased pulmonary inflammation and injury in a rat model of acute lung injury caused by acute necrotizing pancreatitis and may represent a novel therapeutic strategy for the acute necrotizing pancreatitis-associated lung injury.

3-phenyl-propenal is one of the principle compounds isolated from Guizhi (Ramulus Cinnamomi), the principal drug in Guizhi-Tang (GZT), a famous traditional Chinese medical formula. The aim of the present study was to investigate the effects of 3-phenyl-propenal on the expression of toll-like receptor 3 (TLR3), TLR4 and the downstream signaling components on Raw264.7 murine microphages. Raw264.7 cells were cultured in RPMI-1640 medium containing LPS (lipopolysaccharide) or poly (I:C) in the presence or absence of 3-phenyl-propenal. After 24-hour incubation, the medium was collected and the amount of TNF-α and IFN-β was measured by ELISA. mRNA expression of TLR3, TLR4, myeloid differentiation factor (MyD88), TRAF-6 (tumor necrosis factor receptor-associated), TRAM (toll-like receptor-associated molecule) and TRIF (TIR domain-containing adaptor inducing IFN-β) were analyzed by real-time PCR with SYBR green dye. Protein expression of TLR3 and TLR4 was analyzed by Western blotting and that of MyD88 and TRAF-6 was analyzed by immunofluorescence assay. The results indicate that LPS increased the expression of TLR4, MyD88, TRAF-6, TRAM and TRIF, but had no influence on TLR3, while poly (I:C) up-regulated the expression of TLR3, MyD88, TRAM and TRIF. 3-phenyl-propenal significantly decreased the expression of LPS-induced TLR4, MyD88, TRAF-6, while possessing no effect on LPS-induced TRAM and TRIF expression in Raw264.7 cells. When cells were stimulated by poly (I:C), 3-phenyl-propenal significantly decreased TLR3 and MyD88 expression. In conclusion, 3-phenyl-propenal blocked the over-expression of TLR3, TLR4, their downstream signaling components MyD88 and TRAF-6, which indicate that it had an antagonistic effect on TLR3 and TLR4.

Human activities and topography are main factors affecting soil nutrient variation. However, the relationships between these factors are both site- and scale-specific. In hilly areas of south-western China, the dominant cropping systems are rice, vegetables, oranges, Chinese red pepper and maize–sweet potato intercropping. In the present study, slope position (valley, low slope, flat slope, middle slope, upper slope, ridge) and aspect (north, east, south, west) were derived to investigate the relationships among cropping system, terrain, and soil nutrients at county scale. Crops were mainly planted at middle or flat slope positions. Rice and orange plants were evenly distributed across the aspects whereas vegetables were mostly planted on the northern aspect. Red pepper and maize–sweet potato plants were mainly grown on the western and southern aspects. Rice sites had higher contents of organic matter and available nitrogen (N) and lower contents of available phosphorus (P) and available potassium (K). For dryland cropping systems, vegetable sites had higher contents of organic matter, available N, and available P. Red pepper sites had higher contents of available K. Contents of organic matter and available N were generally higher at lower landscape positions. Contents of available K were higher at lower and flat slope positions. Contents of available P were higher at higher landscape positions. Contents of organic matter and available N were higher on the northern and eastern, and lower on the western aspects. Contents of available P were higher on the western and lower on the northern aspects. No significant differences were found for available K across the aspects. Classification tree algorithms indicated that relative importance of the variables on soil nutrient variation was in the order: (i) cropping system, (ii) slope position, and (iii) aspect.

The light-induced absorption of barium titanate doped with rhodium (BaTiO3:Rh) is characterized in near-infrared spectral region. It decreases with the wavelength of the probe beam from 700 to 840 nm, and is significantly dependent on the polarization of the probe beam and insensitive to the polarization of the pump beam. The light-induced absorption as high as 0.32 cm(-1) is obtained for ordinary-polarized probe beam tit lambda = 730 nm. The observations are interpreted by the photorefractive two-center or one center with three valence states model.

Conformational analogues of the hydroxamic acid Oxamflatin 1—compounds 3a, 3b and 4—have been synthesised to enable evaluation of the impact of varying the linking section on histone deacetylase inhibition. Preliminary testing indicates treatment of leukaemia cells with each of the analogues leads to significant inhibition of histone deacetylase and reduction in cell growth and proliferation.

Cinnamaldehyde is a principle compound isolated from Guizhi-Tang, which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. The aim of the present study was to investigate the effects of cinnamaldehyde on expression and activity of cyclooxygenase (COX) and prostaglandin E(2) (PGE(2)) in rat cerebral microvascular endothelial cells (RCMEC). RCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor in cytoplasm of the cells. Then cells were incubated in M199 medium containing interleukin (IL)-1beta in the presence or absence of cinnamaldehyde. After incubation, the medium was collected and the amount of PGE(2) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, mRNA expression and activity of COX were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green dye and ELISA respectively. Positive immunostaining for von Willebrand factor was present diffusely in the cytoplasm of >95% RCMEC. IL-1beta increased the mRNA expression and activity of COX-2, and production of PGE(2) in a dose- and time-dependent manner in RCMEC, while mRNA and activity of COX-1 were not significantly altered. Cinnamaldehyde significantly decreased IL-1beta-induced COX-2 activity and PGE(2) production in a dose-dependent manner, while it showed no inhibitory effect on IL-1beta-induced COX-2 mRNA expression in cultured RCMEC. In conclusion, cinnamaldehyde reduces IL-1beta-induced COX-2 activity, but not IL-1beta-induced COX-2 mRNA expression, and consequently inhibits production of PGE(2) in cultured RCMEC.

Increased plasminogen activator inhibitor type 1 (PAI-1) levels are observed in endothelial cells stimulated by tumour necrosis factor alpha (TNFalpha). Thiazolidinediones (TZDs) may inhibit elevated endothelial cell PAI-1 accounting, in part, for the putative atheroprotective effects of TZDs. In an endothelial cell line, Rosiglitazone (RG) and Pioglitazone (PG) inhibited induction of PAI-1 by TNFalpha. The specific peroxisome proliferator-activated receptor gamma (PPARgamma) inhibitor, SR-202, failed to modulate this effect. RG also inhibited the effect of TNFalpha on a reporter gene construct harbouring the proximal PAI-1 promoter and PAI-1 mRNA in cells co-transfected with a dominant-negative PPARgamma construct. RG and PG attenuated TNFalpha-mediated induction of trans-acting factor(s) Nur77/Nurr1 and binding of nuclear proteins (NP) to the cis-acting element (NBRE). SR-202 failed to modulate these effects. The observations suggest TZDs inhibit TNFalpha-mediated PAI-1 induction independent of inducible PPARgamma activation and this may involve in the modulation of Nur77/Nurr1 expression and NP binding to the PAI-1 NBRE.

We propose and demonstrate a new configuration of mutual- coherent-pumped phase conjugation with Ce:BaTiO 3 . Two coherent beams are incident into a crystal and overlap inside the crystal. The two input beams undergo stimulated photorefractive backscatterings in separated regions, in which their corresponding phase conjugations are generated. Then the four waves in the crystal undergo four-wave mixing. The intensity ratio as well as the interference modulation of the two phase conjugations is characterized as a function of the intensity ratio of the input beams. The phase conjugator exhibits a large intensity dynamic range. We theoretically analyze the modulation transfer function of the mutual-coherent-pumped phase conjugator with Ce:BaTiO 3 . The theoretical analysis is in agreement with the experimental results.