Preconcentration of Cinnamic Acid Derivatives in Traditional Chinese Medicines by an Effective Dispersive Liquid-Liquid Microextraction Based on a Hydrophobic Deep Eutectic Solvent.

A dispersive liquid-liquid microextraction based on hydrophobic deep eutectic solvent (hDES) was developed for the extraction and quantification of four cinnamic acid derivatives in traditional Chinese medicines coupled with high-performance liquid chromatography-ultraviolet detection. In this method, a hDES (tetrabutylammonium chloride-hexanoic acid, molar ratio of 1:2) was prepared as the extractant. It only took 15 s to handle multiple samples simultaneously by hand-assisted dispersion. The use of a narrow-bore tube reduced the amount of the hydrophobic extractant with easier recovery. The approach was influenced by several key parameters, including the composition and consumption of the DES, sample phase pH, salt amount, extraction time, and centrifugation time, all of which had been investigated and optimized. Moreover, the formation of the DES was characterized by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Under the optimal conditions, enrichment factors of the target analytes ranged from 135 to 220. Satisfactory linearities (r ≥ 0.9977), detection limits (0.2-0.4 ng/mL), precision (<8.5%), and accuracy (recoveries: 90.0%-104.6%) were obtained. The method has been successfully applied to the simultaneous extraction and preconcentration of four cinnamic acid derivatives in Chinese medicinal samples with rapidness, high efficiency, and convenience.

Comparison of three-phase hollow fiber liquid-phase microextraction based on reverse micelle with conventional two-phase hollow fiber liquid-phase microextraction and their applications for analysis of cinnamic acids in traditional Chinese medicines.

A novel three-phase hollow fiber liquid-phase microextraction was developed based on reverse micelle as extraction solvent and acceptor phase, and compared with conventional two-phase hollow fiber liquid-phase microextraction. Both procedures were used in the extraction and concentration of four cinnamic acids (caffeic acid, p-hydroxycinnamic acid, ferulic acid, and cinnamic acid) in traditional Chinese medicines prior to high-performance liquid chromatography analysis. Parameters affecting the two procedures were investigated and optimized to obtain the optimum enrichment factors. The mechanism of the developed procedure was explored and elucidated by comparison with conventional two-phase hollow fiber liquid-phase microextraction. Under the optimized conditions, the analytes' enrichment factors were between 50 and 118 for the proposed procedure, and 31-96 for conventional two-phase mode. Satisfactory linear ranges (r2  ≥ 0.99), detection limits (0.1-0.6 ng/mL), precisions (<9.2%), and accuracies (recoveries: 80-123.1%) were observed for the two procedures. The results showed that the enrichment capacity of the proposed procedure for the cinnamic acids is better than that of conventional two-phase procedure, and both are eco-friendly, simple, and effective for the enrichment and detection of cinnamic acids in traditional Chinese medicines.

Purification and characterization of a low-temperature hydroxylamine oxidase from heterotrophic nitrifier Acinetobacter sp. Y16.

OBJECTIVE: To purify a low-temperature hydroxylamine oxidase (HAO) from a heterotrophic nitrifying bacterium Acinetobacter sp. Y16 and investigate the enzyme property. METHODS: A HAO was purified by an anion-exchange and gel-filtration chromatography from strain Y16. The purity and molecular mass were determined by RP-HPLC and SDS-PAGE. The HAO activity was detected by monitoring the reduction of potassium ferricyanide using hydroxylamine as substrate and ferricyanide as electron acceptor. The partial amino acid sequence was determined by mass spectrometry. RESULTS: The low-temperature HAO with a molecular mass of 61 kDa was purified from strain Y16 by an anion-exchange and gel-filtration chromatography. The enzyme exhibited an ability to oxidize hydroxylamine in wide temperature range (4-40 °C) in vitro using hydroxylamine as substrate and ferricyanide as electron acceptor. It was stable in the temperature range of 4 to 15 °C and pH range of 6.0 to 8.5 with less than 30% change in its activity. The optimal temperature and pH were 15 °C and 7.5, respectively. Three peptides were determined by mass spectrometry which were shown to be not identical to other reported HAOs. CONCLUSION: This is the first study to purify a low-temperature HAO from a heterotrophic nitrifier Acinetobacter sp. It differs from other reported HAOs in molecular mass and enzyme properties. The findings of the present study have suggested that the strain Y16 passes through a hydroxylamine-oxidizing process catalyzed by a low-temperature HAO for ammonium removal.

Extragastrointestinal stromal tumors with diffuse membranous distribution with bleeding: A case report.

BACKGROUND: Extragastrointestinal stromal tumors (EGIST) and gastrointestinal stromal tumors are of similar pathological type and form. Here we report a rare case of EGIST diffusely distributed in membranous tissue in abdominal cavity, the feature of which included diffuse tumors at membranous tissue in entire abdominal cavity and spontaneous bleeding of the tumors. CASE SUMMARY: The patient was a 71-year man and hospitalized due to continuous pain at lower abdomen for more than 10 days. Upon physical examination, the patient had flat and tough abdomen with mild pressing pain at lower abdomen, no obvious abdominal mass was touchable, and shifting dullness was positive. Positron emission tomography-computed tomography (CT) showed that in his peritoneal cavity, there were multiple nodules of various sizes, seroperitoneum, multiple enlarged lymph nodes in abdominal/pelvic cavity and right external ilium as well as pulmonary nodules. Plain CT scanning at epigastrium/hypogastrium/pelvic cavity + enhanced three-dimensional reconstruction revealed multiple soft tissue nodules in abdominal/pelvic cavity, peritoneum and right groin. Tumor marker of carbohydrate antigen 125 was 808 U/mL, diffuse tuberous tumor was seen in abdominal/pelvic cavity during operation with hematocelia, and postoperative pathological examination confirmed EGIST. Imatinib was administered with better therapeutic effect. CONCLUSION: Gene testing showed breast cancer susceptibility gene 1 interacting protein C-terminal helicase 1 and KIT genovariation, and the patient was treated with imatinib follow-up visit found that his clinical symptoms disappeared and the tumor load alleviated obviously via imageological examination.

A single-nucleotide polymorphism of the TNFAIP3 gene is associated with systemic lupus erythematosus in Chinese Han population.

Systemic lupus erythematosus (SLE) is a complex systemic disease influenced by genetic and environmental factors. The exact pathogenesis of SLE is still unknown. Recently, several genome-wide association studies (GWA) in European population have found many novel susceptibility genes for SLE including TNFAIP3. In order to examine whether TNFAIP3 is associated with SLE in Chinese Han population, we genotyped one of its non-synonymous mutation SNP rs2230926, showing significant association evidence with SLE in European population, with 1,420 cases and 4,461 controls of Chinese Han by using Sequenom MassArray system. Highly significant association between SNP rs2230926 and SLE of Chinese Han was detected [OR = 1.65, 95% confidence interval (CI): 1.392-1.986, P = 2.03 x 10(-8)]. Interestingly, rs2230926 of TNFAIP3 was also associated with arthritis, ANA and some other subphenotypes of the disease. Our findings suggest that SNP rs2230926 in the TNFAIP3 might be a common genetic factor for SLE within different populations in terms of Chinese Han and European population.

Insonation of Systemically Delivered Cisplatin-Loaded Microbubbles Significantly Attenuates Nephrotoxicity of Chemotherapy in Experimental Models of Head and Neck Cancer.

The use of cisplatin (CDDP), the most common chemotherapy drug for head and neck cancer, is limited by its undesirable side effects, especially nephrotoxicity. We investigated ultrasound microbubbles (USMB) as a tool to increase the local intra-tumoral CDDP level while decreasing systemic CDDP cytotoxicity. We allowed CDDP to interact with human serum albumin and then sonicated the resulting CDDP‒albumin complex to generate CDDP-loaded MBs (CDDP-MBs). We then established a head-and-neck tumor-bearing mouse model by implanting FaDu-fLuc/GFP cells into severe combined immunodeficiency mice and used IVIS® bioluminescence imaging to determine the tumor xenograft formation and size. Twice weekly (until Day 33), we administered CDDP only, CDDP + MBs + US, CDDP-MBs, or CDDP-MBs + US intravenously by tail-vein injection. The US treatment was administered at the tumor site immediately after injection. The in vivo systemic distribution of CDDP indicated that the kidney was the most vulnerable organ, followed by the liver, and then the inner ear. However, CDDP uptake into the kidney and liver was significantly decreased in both the CDDP-MBs and CDDP-MBs + US groups, suggesting that MB binding significantly reduced the systemic toxicity of CDDP. The CDDP-MBs + US treatment reduced the tumor size as effectively as conventional CDDP-only chemotherapy. Therefore, the combination of CDDP-MBs with ultrasound is effective and significantly attenuates CDDP-associated nephrotoxicity, indicating a promising clinical potential for this approach.

Increased oxidative stress and oxidative damage associated with chronic bacterial prostatitis.

AIM: To investigate whether chronic bacterial prostatitis might increase oxidative stress and oxidative damage in chronic bacterial prostatitis patients (CBPP), and to explore its possible mechanism. METHODS: Enrolled in a case-control study were 70 randomly sampled CBPP and 70 randomly sampled healthy adult volunteers (HAV), on whom plasma nitric oxide (NO), vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) level, erythrocyte malondialdehyde (MDA) level, as well as erythrocyte superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities were determined by spectrophotometry. RESULTS: Compared with the HAV group, values of plasma NO and erythrocyte MDA in the CBPP group were significantly increased (P < 0.001); those of plasma VC, VE and beta-CAR as well as erythrocyte SOD, CAT and GPX activities in the CBPP group were significantly decreased (P < 0.001). Findings from partial correlation for the 70 CBPP showed that with prolonged course of disease, values of NO and MDA were gradually increased (P < 0.001), and those of VC, VE, beta-CAR, SOD, CAT and GPX were gradually decreased (P < 0.05-0.001). The findings from stepwise regression for the 70 CBPP suggested that the model was Y = -13.2077 + 0.1894MDA + 0.0415NO - 0.1999GPX, F = 18.2047, P < 0.001, r = 0.6729, P < 0.001. CONCLUSION: The findings suggest that there exist increased oxidative stress and oxidative damage induced by chronic bacterial prostatitis in the patients, and such phenomenon was closely related to the course of disease.

Increased oxidative stress and damage in patients with chronic bacterial prostatitis.

OBJECTIVE: To investigate whether chronic bacterial prostatitis (CBP) increases oxidative stress and damage in patients with CBP, and to explore its possible mechanism. METHODS: Eighty patients with CBP and 80 healthy adults as controls were enrolled in a case-control study, in which levels of nitric oxide (NO), vitamin C (VC), and vitamin E (VE) in plasma, as well as malondialdehyde (MDA), activities of superoxide dismutase (SOD), and catalase (CAT) in erythrocytes were determined by spectrophotometry. RESULTS: Compared with the average values of NO, VC, VE, MDA, SOD, and CAT in the healthy control group, those of plasma NO and erythrocyte MDA in the CBP group were significantly increased (P < 0.001), and those of plasma VC and VE as well as erythrocyte SOD and CAT in the CBP group were significantly decreased (P < 0.001). Findings from partial correlation analysis for course of the disease and NO, VC, VE, MDA, SOD, and CAT in 80 patients with CBP, adjusted for age, suggested that with prolonged course of the disease, values of NO and MDA were gradually increased (P < 0.001), and those of VC, VE, SOD, and CAT were gradually decreased (P < 0.05-0.001). The findings from stepwise regression analysis for course of the disease and NO, VC, VE, MDA, SOD, and CAT in CBP group suggested that the model of stepwise regression was Y = -19.1160 + 0.3112MDA + 0.0337NO, F = 22.1734, P < 0.001, r = 0.6045, P < 0.001. The findings from the reliability analysis for VC, VE, SOD, CAT, NO, and MDA in the CBP group showed that the reliability coefficients' alpha (6 items) was 0.7195, P < 0.0001, and the standardized item alpha was 0.9307, P < 0.0001. CONCLUSION: There exist increased oxidative stress and damage induced by chronic bacterial prostatitis in patients, and such a phenomenon is closely related to the course of disease.

Overweight and obesity-induced oxidative stress in children.

OBJECTIVE: To investigate whether overweight and obesity might cause oxidative stress and potential oxidative damage in overweight and obese children, and to explore its possible mechanism. METHODS: Eighty-five overweight and obese children (OOC), and eighty-five age-matched healthy children (HC) were recruited in this case-control study. The present study analyzed spectrophotometrically vitamin C (VC), vitamin E (VE), and 3-carotene (P-CAR) in plasma, as well as the activities of superoxide dismutase (SOD), catalase (CAT), and the level of malondialdehyde (MDA) in erythrocytes. RESULTS: Compared with those of VC, VE, P-CAR, SOD, CAT and MDA in the HC group, the average values of VC, VE, 3-CAR, SOD, and CAT in the OOC group were significantly decreased (P<0.001), while the average value of MDA in the OOC group was significantly increased (P<0.001). The regression analysis demonstrated that VC, VE, P-CAR, SOD, and CAT were negatively correlated (P<0.05-0.01), and MDA was positively correlated with BMI (P<0.05). Fitting to the model of multiple stepwise regression of BMI on VC, VE, P-CAR, SOD, CAT, and MDA in 85 OOC was Y= 27.0041 + 0.2541MDA - 2.1448beta-CAR - 0.0090CAT, where F= 43.8088, P<0.001, r = 0.7866, r(2)= 0.6187, adjusted r(2)= 0.6046. The findings from the reliability analysis for VC, VE, P-CAR, SOD, CAT, and MDA used to reflect increased oxidative stress and potential oxidative damage in the OOC showed that the reliability coefficients (alpha, 6 items) = 0.7231, P<0.0001, and that the standardized item alpha = 0.9207, P<0.0001. CONCLUSION: The present study suggests that there exists an increased oxidative stress in overweight and obese children.

Increased oxidative stress in women with pregnancy-induced hypertension.

OBJECTIVE: To investigate whether pregnancy-induced hypertension (PIH) may increase oxidative stress in women with PIH, and to explore the mechanisms by which PIH may increase oxidative stress and potential free radical damage. METHODS: Seventy women with PIH and seventy women with uncomplicated normotensive pregnancy (UNP) whose age, nutritional conditions, levels of hemoglobin and albumin were all matched, were enrolled in a randomized controlled trial. Their plasma concentrations of nitric oxide (NO), vitamin C (VC), vitamin E (VE), and beta-carotene (beta-CAR) as well as their erythrocyte malondialdehyde (MDA), and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) were determined by spectrophotometry. RESULTS: Compared with average values of the above experimental parameters in the women with UNP, the average value of erythrocyte MDA in the women with PIH significantly increased (P<0.0001), while the average values of plasma NO, VC, VE, and beta-CAR as well as those of erythrocyte SOD, CAT, and GPX in the women with PIH significantly decreased (P<0.0005-0.0001). The findings from partial correlation analysis (controlling for age) for 70 women with PIH showed that with elevated systolic blood pressure (SBP) and diastolic blood pressure (DBP), MDA value gradually increased (P<0.001), and NO, VC, VE, beta-CAR, SOD, CAT, and GPX values gradually decreased (P<0.02-0.001). The findings from reliability analysis for NO, VC, VE, beta-CAR, SOD, CAT, GPX, and MDA values used to reflect increased oxidative stress and potential free radical damage in women with PIH showed that the reliability coefficients (alpha, 8 items) = 0.7062, P<0.0001, and the standardized item alpha = 0.9116, P<0.0001. CONCLUSION: The findings in the present research suggest that pregnancy-induced hypertension can increase oxidative stress and potential free radical damage in women with pregnancy-induced hypertension.

Oxidative stress and free radical damage in patients with acute dipterex poisoning.

OBJECTIVE: To investigate whether acute dipterex poisoning (ADP) may cause oxidative stress and free radical damage in the bodies of acute dipterex poisoning patients (ADPPs), and to explore the mechanisms by which ADP may cause oxidative stress and free radical damage. METHODS: Fifty ADPPs and fifty healthy adult volunteers (HAVs) whose ages, gender and others were matched with the ADPPs were enrolled in a randomized controlled study, in which concentrations of nitric oxide (NO), vitamin C (VC), vitamin E (VE) and beta-carotene (beta-CAR) in plasma as well as concentration of lipoperoxide (LPO), and activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and acetylcholinesterase (AChE) in erythrocytes were determined by spectrophotometric analytical methods. RESULTS: Compared with the average values of experimental parameters in the HAVs group, the average values of plasma NO and erythrocyte LPO in the ADPPs group were significantly increased (P<0.0001), while those of plasma VC, VE and beta-CAR as well as erythrocyte SOD, CAT, GPX and AChE in the ADPPs group were significantly decreased (P<0.0001). Bivariate correlation analysis and partial correlation analysis suggested that when NO and LPO values were increased, and VC, VE, beta-CAR, SOD, CAT and GPX values were decreased in the ADPPs, AChE value was decreased gradually in the ADPPs (P<0.001-0.0001). Reliability analysis of experimental parameters reflecting oxidative stress and free radical damage in the ADPPs showed that the reliability coefficient (8 items) alpha=0.6909, and the standardized item alpha=0.8574. CONCLUSION: The findings in the present study suggest that ADP can cause oxidative stress and free radical damage, and inhibit markedly erythrocyte acetylcholinesterase activity in ADPPs.

Genome-wide association study identifies two new susceptibility loci for atopic dermatitis in the Chinese Han population.

Atopic dermatitis is a chronic, relapsing form of inflammatory skin disorder that is affected by genetic and environmental factors. We performed a genome-wide association study of atopic dermatitis in a Chinese Han population using 1,012 affected individuals (cases) and 1,362 controls followed by a replication study in an additional 3,624 cases and 12,197 controls of Chinese Han ethnicity, as well as 1,806 cases and 3,256 controls from Germany. We identified previously undescribed susceptibility loci at 5q22.1 (TMEM232 and SLC25A46, rs7701890, P(combined) = 3.15 × 10(-9), odds ratio (OR) = 1.24) and 20q13.33 (TNFRSF6B and ZGPAT, rs6010620, P(combined) = 3.0 × 10(-8), OR = 1.17) and replicated another previously reported locus at 1q21.3 (FLG, rs3126085, P(combined) = 5.90 × 10(-12), OR = 0.82) in the Chinese sample. The 20q13.33 locus also showed evidence for association in the German sample (rs6010620, P = 2.87 × 10(-5), OR = 1.25). Our study identifies new genetic susceptibility factors and suggests previously unidentified biological pathways in atopic dermatitis.

Association analyses identify six new psoriasis susceptibility loci in the Chinese population.

We extended our previous genome-wide association study for psoriasis with a multistage replication study including 8,312 individuals with psoriasis (cases) and 12,919 controls from China as well as 3,293 cases and 4,188 controls from Germany and the United States and 254 nuclear families from the United States. We identified six new susceptibility loci associated with psoriasis in the Chinese study containing the candidate genes ERAP1, PTTG1, CSMD1, GJB2, SERPINB8 and ZNF816A (combined P < 5 × 10⁻8) and replicated one locus, 5q33.1 (TNIP1-ANXA6), previously reported (combined P = 3.8 × 10⁻²¹) in the European studies. Two of these loci showed evidence for association in the German study at ZNF816A and GJB2 with P = 3.6 × 10⁻³ and P = 7.9 × 10⁻³, respectively. ERAP1 and ZNF816A were associated with type 1 (early onset) psoriasis in the Chinese Han population (test for heterogeneity P = 6.5 × 10⁻³ and P = 1.5 × 10⁻³, respectively). Comparisons with the results of previous GWAS of psoriasis highlight the heterogeneity of disease susceptibility between the Chinese and European populations. Our study identifies new genetic susceptibility factors and suggests new biological pathways in psoriasis.

Genome-wide association study for vitiligo identifies susceptibility loci at 6q27 and the MHC.

We conducted a genome-wide association study of generalized vitiligo in the Chinese Han population by genotyping 1,117 cases and 1,429 controls. The 34 most promising SNPs were carried forward for replication in samples from individuals of the Chinese Han (5,910 cases and 9,916 controls) and Chinese Uygur (713 cases and 824 controls) populations. We identified two independent association signals within the major histocompatibility complex (MHC) region (rs11966200, Pcombined=1.48x10(-48), OR=1.90; rs9468925, Pcombined=2.21x10(-33), OR=0.74). Further analyses suggested that the strong association at rs11966200 might reflect the reported association of the HLA-A*3001, HLA-B*1302, HLA-C*0602 and HLA-DRB1*0701 alleles and that the association at rs9468925 might represent a previously unknown HLA susceptibility allele. We also identified one previously undescribed risk locus at 6q27 (rs2236313, Pcombined=9.72x10(-17), OR=1.20), which contains three genes: RNASET2, FGFR1OP and CCR6. Our study provides new insights into the genetic basis of vitiligo.

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29.

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC(50)) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 micromol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.

A novel KIT missense mutation in one Chinese family with piebaldism.

Piebaldism is an autosomal dominant disorder characterized by congenital leukoderma, mostly affecting forehead, abdomen and knee. Previous studies have revealed that piebaldism is caused by mutations of the KIT gene, which encodes the cell surface transmembrane tyrosine kinase receptor for KIT ligand. We reported here a Chinese Han family with piebaldism, and performed mutation detection of KIT gene by direct sequencing. A novel missense mutation C58G was identified in the patients, but not in the healthy individuals from the family and 100 unrelated controls. This study contributes to the database on KIT in piebaldism and enriches the knowledge about the genotype/phenotype correlation.

Psoriasis genome-wide association study identifies susceptibility variants within LCE gene cluster at 1q21.

We report the first large genome-wide association study (GWAS) in a Chinese population to identify susceptibility variants for psoriasis using a two-stage case-control design. In the first stage, we carried out a genome-wide association analysis in 1,139 cases and 1,132 controls of Chinese Han ancestry using Illumina Human 610-Quad BeadChips. In the second stage, we took top SNPs forward for replication in two independent samples of 5,182 cases and 6,516 controls of Chinese Han ancestry, and 539 cases and 824 controls of Chinese Uygur ancestry. In addition to the strong replication for two known susceptibility loci MHC (rs1265181, P = 1.93 x 10(-208), OR = 22.62) and IL12B (rs3213094, P(combined) = 2.58 x 10(-26), OR = 0.78), we identified a new susceptibility locus within the LCE gene cluster on 1q21 (rs4085613, P(combined) = 6.69 x 10(-30), OR = 0.76).