Quantification of soluble epoxide hydrolase inhibitors in experimental and clinical samples using the nanobody-based ELISA

To ensure proper dosage of a drug,analytical quantification of it in biofluid is necessary.Liquid chromatography mass spectrometry(LC-MS)is the conventional method of choice as it permits accurate identification and quantification.However,it requires expensive instrumentation and is not appropriate for bedside use.Using soluble epoxide hydrolase(sEH)inhibitors(EC5026 and TPPU)as examples,we report development of a nanobody-based enzyme-linked immunosorbent assay(ELISA)for such small molecules and its use to accurately quantify the drug chemicals in human samples.Under optimized conditions,two nanobody-based ELISAs were successfully established for EC5026 and TPPU with low limits of detection of 0.085 ng/mL and 0.31 ng/mL,respectively,and two order of magnitude linear ranges with high precision and accuracy.The assay was designed to detect parent and two biologically active metabolites in the investigation of a new drug candidate EC5026.In addition,the ELISAs displayed excellent correlation with LC-MS analysis and evaluation of inhibitory potency.The results indicate that nanobody-based ELISA methods can efficiently analyze drug like compounds.These methods could be easily implemented by the bedside,in the field in remote areas or in veterinary practice.This work illustrates that nanobody based assays offer alternative and supplementary analytical tools to mass spectrometry for monitoring small molecule medicines during clinical development and therapy.Attributes of nanobody based pharmaceutical assays are discussed.

Eukaryotic expression, purification and activity characterization of human soluble DSG2 extracellular domain protein

Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.

Elevated soluble fas blood concentrations in patients dying from spontaneous intracerebral hemorrhage

BACKGROUND Several studies of spontaneous intracerebral hemorrhage(SICH)patients have shown apoptotic changes in brain samples after hematoma evacuation.However,there have been no data on the association between blood concentrations of soluble fas(sFas)(the main surface death receptor of the extrinsic apoptosis pathway)and the prognosis of spontaneous intracranial hypotension(SIH)patients.AIM To determine whether there is an association between blood sFas concentrations and SICH patient mortality.METHODS We included patients with severe and supratentorial SIH.Severe was defined as having Glasgow Coma Scale<9.We determined serum sFas concentrations at the time of severe SICH diagnosis.RESULTS We found that non-surviving patients(n=36)compared to surviving patients(n=39)had higher ICH score(P=0.001),higher midline shift(P=0.004),higher serum sFas concentrations(P<0.001),and lower rate of early hematoma evacuation(P=0.04).Multiple logistic regression analysis showed an association between serum sFas concentrations and 30-d mortality(odds ratio=1.070;95%confidence interval=1.014-1.129;P=0.01)controlling for ICH score,midline shift,and early hematoma evacuation.CONCLUSION The association of blood sFas concentrations and SICH patient mortality is a novel finding in our study.

Experimental verification of nanonization enhanced solubility for poorly soluble optoelectronic molecules

Solubility enhancement has been a priority to overcome poor solubility with optoelectronic molecules for solution-processable devices. This study aims to obtain experimental data on the effect of particle sizes on the solubility properties of several typical optoelectronic molecules in organic solvents, including the solubility results of 1,3-bis(9-carbazolyl)benzene(m CP), 1,3,5-tris(1-phenyl-1H-benzimidazol-2-yl)ben zene(TPBi) and 2-(4-tert-butylphenyl)-5-(4-biphenyl)-1,3,4-oxadiazole(PBD) in ethanol and acetonitrile,respectively. Nanoparticles of m CP, TPBi and PBD with sizes from dozens to several hundred nanometers were prepared by solvent antisolvent precipitation method and their solubility were determined by using isothermal saturation method. The saturation solubility of nanoparticles of three kinds of optoelectronic molecules exhibited increase of 12.9%-25.7% in comparison to the same raw materials in the form of microparticles. The experimental evidence indicates that nanonization technology is a feasible way to make optoelectronic molecules dissolve in liquids with enhanced solubility.

Modifi cation of wheat bran insoluble and soluble dietary fibers with snail enzyme

Insoluble dietary fiber(IDF)and soluble dietary fiber(SDF)extracted from wheat bran were modifi ed by snail enzyme and their physicochemical properties(water retention capacity and oil retention capacity),functional properties(cholesterol adsorption capacity,glucose adsorption capacity and antioxidant activity)and structural characterizations were evaluated.The results showed that snail enzyme modification led to the significant increase in oil retention capacity of IDF,glucose adsorption capacity and cholesterol adsorption capacity of IDF and SDF.Enzymatic modification also markedly improved the DPPH radical scavenging capacity and reducing power of IDF and SDF.Meanwhile,scanning electron microscopy(SEM)analysis indicated the microstructures of IDF and SDF powders were signifi cantly changed.Fourier transfer-infrared spectrometry(FT-IR)showed that snail enzyme modifi cation could degrade the part of cellulose and hemicellulose of IDF and SDF.All these improved physicochemical and functional properties of IDF and SDF might depend on their structural changes.It suggested that snail enzyme modifi cation could effectively improve physicochemical and functional properties of IDF and SDF from wheat bran.

The Role of Soluble, Insoluble Fibers and Their Bioactive Compounds in Cancer: A Mini Review

The cancer incidence has risen dramatically over the last decades. About 8 million people died globally according to latest reports, which represented almost 40% more than it was 20 years ago. Risk factors for the development of cancer have been found to include smoking, alcohol, drugs, obesity and diet. Fiber intake has shown to exhibit chemoprotective effects on cancer proliferation and metastasis that may seem to be very promising. This article will review the role of different types of fiber such as, cellulose, lignin, pectin and inulin in development and prevention of different types of cancers. This article would also discuss the effectiveness of both types of fiber in cancer prevention.

Solvent de binding of water soluble binder in powder injection moulding

The solvent debinding of water soluble binder in powder injection moulding (MIM) was investigated systematically, including the effects of solvent types, temperature and the thickness of green parts on the solvent debinding rate. After studying the debinding of a green part with a thickness of 4.26 mm, it was found that, the debinding rate of polyethylene glycol(PEG) in water and alcohol was high initially, and then decreased; however, it would increase with temperature increasing. At room temperature, the dissolution rate of PEG in water was higher than that in alcohol, but the latter would be much faster with temperature increasing because the debinding activation energy in alcohol was 51.44 kJ·mol -1 ·K -1 , much higher than 24.23 kJ·mol -1 ·K -1 in water. With a green part thickness larger than 4.26 mm, the debinding was controlled by diffusion; but with that smaller than 2.36 mm, the debinding was controlled by both dissolution and diffusion.

Effect of Alkali Stress on Soluble Sugar, Antioxidant Enzymes and Yield of Oat

Alkali stress can cause severe crop damage and reduce production. However, physiological processes involved in alkali stress in oat seedlings are not well understood. In this study, physiological responses and yield of oat to alkali stress were studied using the alkali-tolerant oat genotype Vao-9 and the alkali-sensitive oat genotype Baiyan 5. The results were： （i） low concentrations of alkali stress （25 and 50 mmol L^-1） significantly reduced the yield and grain weight while increased the oat grain number per spike. A negative correlation between yield and malondialdehyde （MDA） content at the jointing and grain filling stages and positive correlations between yield on one hand and superoxide dismutase （SOD）, and peroxidase （POD） activities on the other at the jointing stage were observed. There was a positive correlation between MDA and soluble sugar at the grain filling stage; （ii） soluble sugar content was increased at the jointing and grain filling stages and decreased at the heading stage by alkali stress; （iii） alkali stress increased the SOD activity during the heading and grain filling stages, and increased the POD activity at the heading stage. As compared to the control, the increase of MDA contents in alkali-treated oat was observed, during the jointing, heading and grain filling stages; （iv） under alkali stress, the oat genotype Vao-9 showed higher antioxidant enzyme activity and lower soluble sugar contents during the heading stage, and lower MDA contents than those in the oat genotype Baiyan 5 under alkali stress. The result suggested that the high ROS scavenging capacity and soluble sugar levels might play roles in oat response to alkali stress,

Effects of operational factors on soluble microbial products in a carrier anaerobic baffled reactor treating dilute wastewater

The effects of feed strength, hydraulic residence time （HRT）, and operational temperatures on soluble microbial product （SMP） production were investigated, to gain insights into the production mechanism. A carrier anaerobic baffled reactor （CABR） treating dilute wastewater was operated under a wide range of operational conditions, namely, feed strengths of 300-600 mg/L, HRTs of 9- 18 h, and temperatures of 10-28℃. Generally, SMP production increased with increasing feed strength and decreasing temperature. At high temperature （28℃）, SMP production increased with decreasing HRT. As the temperature was decreased to 18 and 10℃, the SMP production was at its peak for 12 h HRT. Therefore, temperature could be an important determinant of SMP production along with HRT. A higher SMP to soluble chemical oxygen demand （SCOD） ratio was found at high temperature and long HRT because of complete volatile fatty acid degradation. SMP accounted for 50%-75% of the SCOD in the last chamber of the CABR. As a secondary metabolite, some SMP could be consumed at lower feed strength.

Changes of Oxidative Stress and Soluble Sugar in Anthers Involve in Rice Pollen Abortion Under Drought Stress

Two rice maintaining lines with different drought tolerances, viz., Jin 23B （tolerant） and Zhenshan 97B （sensitive）, were used to study the oxidative stress and soluble sugar in rice anthers and pollen viability under drought stress during flowering stage. Higher antioxidant enzyme activities and lower malonaldehyde （MDA） content in rice anthers were observed in Jin 23B than in Zhenshan 97B under drought stress. Further, a great increase in the content of soluble sugar in rice anthers of Jin 23B was observed across the whole drought exposure, while Zhenshan 97B showed significant decrease in soluble sugar during 9-12 d after drought stress （DADS）. Accordingly, a marked decline of pollen fertility and activity, pollen numbers in an anther and pollen numbers on a stigma was observed in Zhenshan 97B, whereas little difference was found in Jin 23B. Thus, we suggest that pollen abortion caused by drought stress may be related with the reciprocity between oxidative stress and soluble sugar content in rice anthers.

Soluble protein and acid phosphatase exuded by ectomycorrhizal fungi and seedlings in response to excessive Cu and Cd

Fungi and their symbionts can alleviate heavy metal stress by exuding soluble proteins and enzymes. This study examined the role of soluble protein and acid phosphatase （APase） exuded by Xerocomus chrysenteron, an ectomycorrhizal fungus, and the seedlings of its symbiont, Chinese pine （Pinus tabulaeformis）, under conditions of excessive Cu and Cd. The growth type showed that this poorly studied ectomycorrhizal fungus was capable of tolerating high concentrations of Cu, and may be useful in phytoremediation. X. chrysenteron grew well at 80 mg/L Cu, and the EC50 for Cd was 17.82 mg/L. X. chrysenteron also showed enhanced exudation of soluble protein in both isolated and inoculated cultivations under the influence of Cu and Cd. Soluble protein exudation, however, differed under Cu and Cd stress in isolates. In mediums containing Cu, soluble protein exudation increased with concentration, but in mediums containing Cd the content of soluble protein increased to a comparable level at all concentrations. This study demonstrated that soluble protein was related to heavy metal tolerance, although the different ions played different roles. While APase activity in exudates of fungi and seedlings decreased under Cu and Cd stress in comparison to the control, the APase activity in seedlings was maintained by inoculation. Thus, X. chrysenteron facilitated the ability of plant to maintain a normal nutrient uptake, and therefore to protect it from heavy metal toxicity.

Photocatalytic Degradation of Water-Soluble Dyes by LaCoO_3

Perovskite type oxides LaCoO 3 was prepared by citrate method in granula of 20～30 nm. Using a fluorescent Hg lamp or sunlight as irradiator, the degradation experiments of various water soluble dyes were carried out in the suspension system of LaCoO 3. The results show that the perovskite type oxide LaCoO 3 has good photocatalytic activity. With the study of X ray photoelectron spectroscopy and photoacoustic spectra, its photocatalytic activity is mainly related with the factors such as the d electron structure of ion Co 3+ , Co O binding energy and adsorbed oxygen on the surface etc.

Serum soluble ST2 is a promising prognostic biomarker in HBV-related acute-on-chronic liver failure

BACKGROUND： The IL-33/ST2 axis is involved in the pathogenesis of many diseases such as autoimmune diseases, cancer,and heart failure. However, studies of the IL-33/ST2 pathway in HBV-related acute-on-chronic liver failure（HBV-ACLF） are lacking. The present study aimed to determine the prognostic role of serum IL-33/soluble ST2（s ST2） in HBV-ACLF.METHODS： Serum levels of IL-33 and sS T2 in healthy controls（HC, n=18）, chronic hepatitis B（CHB, n=27） and HBV-ACLF（n=51） patients at the 1st and 4th week after enrollment were detected using ELISA, and clinical data were collected. The follow-up of HBV-ACLF patients lasted for 6 months at least.RESULTS： There was no significant difference of serum IL-33 level among HC, CHB and HBV-ACLF patients at week 1.However, serum s ST2 level differed significantly among the three groups： highest in the HBV-ACLF group, moderate in the CHB group and lowest in the HC group. There was a reverse correlation between serum s ST2 level and the survival of HBV-ACLF patients. The level of serum s ST2 in HBV-ACLF survivors was significantly declined from week 1 to week 4 following the treatment, whereas that in HBV-ACLF nonsurvivors remained at a high level during the same period. Furthermore, serum sS T2 level was significantly correlated with laboratory parameters and the most updated prognostic scores（CLIF-C OF score, CLIF-C ACLF score and ACLF grades）. Thereceiver operating characteristics curves demonstrated that serum sS T2 level was a good diagnostic marker for predicting the 6-month mortality in HBV-ACLF patients, comparable to the most updated prognostic scores. Serum sS T2 cut-off points for predicting prognosis in HBV-ACLF patients were 76 ng/mL at week 1 or 53 ng/mL at week 4, respectively. HBV-ACLF patients with serum sS T2 level above the cut-off point often had a worse prognosis than those below the cut-off point.CONCLUSION： Serum s ST2 may act as a promising biomarker to assess severity and predict prognosis of patients with HBV-ACLF and help for the early identification and optimal treatment of HBV-ACLF patients at high risk of mortality.

Effectiveness of fluidized pellet bed for removing soluble contaminants

Fluidized pellet bed （FPB） has been successfully applied in water and wastewater treatment. However, the removal mechanism of contaminants especially the soluble ones, is still unclear. This study aimed to evaluate the effectiveness of FPB reactor for removing soluble contaminants from synthetic wastewater. By only coagulation through jar test operation with addition of polyaluminium chloride （PAC1） as primary coagulant and polyacryamide （PAM） as coagulant-aid, the removals of soluble chemical oxygen demand （COD）, total phosphorus （TP）, and NH4^＋-N were found to be only 2.2%-7.5%, 5.7%-25.5%, and 9.9%-18.5%, respectively. However, by FPB operation under the same dosage of coagulants, these values increased to 82.7%, 37.2%, and 50%, indicating that the formation of pellets in the FPB effectively enhanced the removal of soluble contaminants. By careful comparison of the settleablility and filterability of the pollutants after coagulation, the originally soluble contaminants could be divided into three groups, namely： （1） coagulated-and- settleable, （2） coagulated-but-nonsettleable, and （3） uncoagulable. It was found that not only the first two groups but also a large part of the third group could be effectively removed by FPB operation. However, the mechanism for the removal of the uncoagulable pollutants by FPB operation still needs further investigation.

Relationship Between Soluble Protein,Chlorophyll and ATP in Drought-Resistant Sweet Potato Under Water Stress

Some indices concerning the metabolism of substance and energy in sweet potato leaves under water stress were studied. The results showed an obvious increase in soluble protein content. Compared with control, Chl a, Chl b, total Chl contents and the ratio of Chl a to Chl b all decreased to some extent. ATP content increased in some varieties and decreased in others, but the stronger the drought resistance of the variety , the higher the ATP content. The correlation coefficient(r)of the soluble protein content, ratio of Chl a to Chl b and ATP content as a percentage of the drought-resistant sweet potato control variety are 0. 8968, - 0. 8509 and 0. 8200, respectively, P<0. 01. So these indices can be used to evaluate the drought resistance of different sweet potato varieties.

Changes in soluble interleukin-2 receptor level in serum and Na^+ -K^+ -exchanging ATPase activity in semen of infertile men caused by antisperm antibody

Aim: To explore the possible mechanisms of male infertility caused by antisperm antibody (AsAb). Methods: Thesoluble interleukin-2 receptor (sIL-2R) level in serum was analyzed by ELISA and Na^+ -K^+ -exchanging ATPase activi-ty in semen by phosphorus (Pi) assay. Results: The slL-2R level in serum was significantly higher and the Na^+ -K^+ -exchanging ATPase activity in semen significantly lower in AsAb positive infertile men when compared with thecontrols. Conclusion: The AsAb titer varies with the slL-2R level in serum. A decrease in Na^+ -K^+ -exchangingATPase activity in semen may play a role in male infertility caused by AsAb.

Biodegradability of soil water soluble organic carbon extracted from seven different soils

Water soluble organic carbon （WSOC） is considered the most mobile and reactive soil carbon source and its characterization is an important issue for soil ecology study. A biodegradability test was set up to study WSOC extracted from 7 soils differently managed. WSOC was extracted from soil with water （soil/water ratio of 1：2, W/V） for 30 min, and then tested for biodegradability by a liquid state respirometric test. Result obtained confirmed the finding that WSOC biodegradability depended on the both land use and management practice. These results suggested the biodegradability test as suitable method to characterize WSOC, and provided useful information to soil fertility.

Effect of Cadmium and Herbicides on the Growth, Chlorophyll and Soluble Sugar Content in Rice Seedlings

To investigate the combined effects of Cd^2＋ （ 0,10,100,500 μmol/L） , acetochlor （AC） （0,1.6,4.0,8.0μmol/L）, and bensulfuronmethyl （BSM） （ 0,0.16,0.40,0.80 μmol/L） on the biomass,leaf soluble sugar, total chlorophyll （chl） content, chl a/b ratio, and Cd content in roots and shoots in the rice seedlings, pot culture experiments were conducted with rice （Oryza sativa L. ） cultivar Jinyou 402. The results showed that the Cd and AC combined treatment （Cd＋ AC treatment） significantly inhibited the growth of the roots and shoots. The root dry weight/shoot dry weight （RDW/SDW） ratio, total chlorophyll content and chl a/b ratio decreased by 41%, 50%, 56% , respectively, in comparison with the control, as well as the leaf soluble sugar content, plant dry weight /plant fresh weight （PDW/PFW） ratio increased by 284% and 44% , respectively. Cd content in the roots and shoots had a decreasing tendency with the increasing concentration of AC under the Cd＋AC treatment. The Cd and BSM combined treatment （Cd＋BSM treatment） had a similar result to the Cd＋AC treatment. The results suggest that the toxicity of Cd to rice seedlings has been enhanced due to its combination with herbicides.

Study on the Rapid Method to Predict Longevity of Controlled Release Fertilizer Coated by Water Soluble Resin

The study discussed the rapid method to test and predict the longevity of controlled release fertilizers （CRFs） coated by water soluble resin by using the short-term leaching under higher temperature. Pure water dissolving incubation and higher temperature leaching were used to study the patterns of the nutrient release of the CRFs. The correlation analysis between the days at 25℃ and the hours at 80℃ of Trincote 1 and Trincote 2 for the same cumulative release rates were conducted. Patterns of cumulative nutrient release curve followed one factor quadratic regression equation at each given temperatures, and each of relative coefficient was bigger than 0.995. As the temperature increased, nutrients release of the CRFs increased. The longevity of resin coated CRFs were predicted by use of both the cumulative nutrients release equation at 80℃ and the regression equation of release time needed for the same cumulative release rates between 25 and 80℃. There were only 0.3-6.9% relative errors between the tested longevity and predicted one. In conclusion, the longevity of resin coated CRFs could be predicted more quickly and precisely by use of the higher temperature short-term leaching method than that of the traditional differential release rate. The longevity of resin coated CRF could be rapidly and precisely predicted in a few hours by application of the higher temperature shortterm leaching method.

Increased serum levels of soluble CD146 and vascular endothelial growth factor receptor 2 in patients with exudative age-related macular degeneration

AIM: To investigate serum levels of soluble CD146(s CD146) and vascular endothelial growth factor receptor 2(VEGFR2) in patients with age-related macular degeneration(AMD). METHODS: Eighty-eight patients with exudative AMD and 45 sex-and age-matched healthy controls were enrolled in this study conducted in China. Serum samples was obtained from the patients with exudative AMD and from the controls. Serum sCD146 and VEGFR2 protein levels were measured using an enzyme-linked immunosorbent assay.RESULTS: We found that serum sCD146 and VEGFR2 protein levels were significantly higher in the patients with exudative AMD group than in the controls(t=3.859, P<0.001 and t=3.829, P<0.001, respectively). Serum sCD146 levels were significantly higher in patients with classic choroidal neovascularization(CNV) than in those with occult CNV(t=9.899, P<0.001). There was a significant difference in the trend for exudative AMD in the highest versus lowest quartile of circulating sCD146 levels(χ2=10.29, P=0.001). The receiver operating characteristic curve analysis showed that the area under the curve was 0.696 for s CD146(95%CI: 0.601-0.791) with an optimum diagnostic cut-off value of 157.16 ng/mL, a sensitivity of 55.7%, and a specificity of 82.2%.CONCLUSION: The serum sCD146 level increases and may be a biomarker for exudative AMD.