Identification and Characterization of Pectate Lyase as a Novel Allergen in Artemisia sieversiana Pollen.

INTRODUCTION: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. METHODS: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen's physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. RESULTS: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients' serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients' basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel ß-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. CONCLUSION: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy.

[Risk Factors Associated with Intraoperative Blood Loss in Patients with Early Cervical Cancer].

OBJECTIVE: To determine risk factors associated with intraoperative blood loss in patients with early cervical cancer (stage ⅠB-ⅡA). METHODS: The medical records of 892 patients who underwent surgical treatments for early cervical cancer in the Second West China University Hospital of from Dec 2010 to Sep 2017 were retrospectively reviewed: 127 having ≥500 mL intraoperative blood loss patients compared with 765 less than 500 mL. Differences between the two groups in age, body mass index (BMI), gravidity, history of abdominal and pelvic operations, chronic pelvic inflammation disease, clinical stage, methods of operation, neoadjuvant chemotherapy (NACT) and post-NACT operative opportunity, preoperative and postoperative hemoglobin, and intraoperative transfusion volume were analyzed through univariate and multivariate statistical methods. RESULTS: The univariate analyses identified age, BMI, gravidity, history of abdominal and pelvic operation, chronic pelvic inflammation disease, clinical stage, methods of operation, NACT and post-NACT operative opportunity assignificant factors associated with intraoperative blood loss ( P<0.05). The multivariate logistic regression analysis confirmed that age ≥40 yr. [partial regression coefficient (B)=2.100)], BMI ≥24 kg/m2 (relative to 18.5-23.9 kg/m2)(B=1.842) , clinical stage ⅡA (relative to phase ⅠB, B=2.401) , trans-abdominal operative method (relative to laparoscopy, B=1.347), no NACT (B=1.540) and post-NACT operative opportunity <2 or >3 weeks (relative to within 2-3 weeks) (B=1.723) were independent predictors of higher intraoperative blood loss (≥500 mL). CONCLUSION: Clinical stage and age, etc. are risk factors associated with intraoperative blood loss in patients with early cervical cancer.

TET1-GPER-PI3K/AKT pathway is involved in insulin-driven endometrial cancer cell proliferation.

Large amount of clinical evidence has demonstrated that insulin resistance is closely related to oncogenesis of endometrial cancer (EC). Despite recent studies showed the up-regulatory role of insulin in G protein-coupled estrogen receptor (GPER/GPR30) expression, GPER expression was not decreased compared to control when insulin receptor was blocked even in insulin treatment. The purpose of this study was to explore the possible mechanism by which insulin up-regulates GPER that drives EC cell proliferation. For this purpose, we first investigated the GPER expression in tissues of endometrial lesions, further explored the effect of GPER on EC cell proliferation in insulin resistance context. Then we analyzed the role of Ten-Eleven Translocation 1 (TET1) in insulin-induced GEPR expression and EC cell proliferation. The results showed that GPER was highly expressed in endometrial atypical hyperplasia and EC tissues. Mechanistically, insulin up-regulated TET1 expression and the latter played an important role in up-regulating GPER expression and activating PI3K/AKT signaling pathway. TET1 mediated GPER up-regulation was another mechanism that insulin promotes EC cell proliferation.

[Research of inhibition of compound gan shu kang on hepatitis B virus].

OBJECTIVE: To study the anti-HBV effect in vitro of each extract from compound Gan Shu Kang. METHODS: The toxicity was investigated with cytopathic effect (CPE) by enzyme linked immuno-adsorbed assay (ELISA), the inhibitory effect of each extract on HBsAg and HBeAg secreted by 2215 cell line infected by HBV DNA was evaluated. RESULTS: The half of the toxic concentration (TC50) of petroleum ether, ethyl acetate and n-butanol was 125, 375 and 62.5 microg/mL, respectively. The maximum nontoxic concentration (TC0) was 62.5, 125 and 31.3 microg/mL, respectively. The medium effective concentration (IC50) of petroleum ether and ethyl acetate on 2215 cellular HBsAg express the inhibitory effect was 48.6 and 14.0 microg/mL, HBeAg was 52.4 and 19.7 microg/mL, while the IC50 of n-butanol for HBsAg and HBeAg was more than 125 microg/mL. The therapeutic index (TI) of petroleum ether was 2.57 and 2.43, ethyl acetate was 26.7 and 19.04, n-butanol was both less than 2. CONCLUSION: The anti-HBV effect of ethyl acetate extract of compound Gan Shu Kang is better than that of petroleum ether, while the n-butanol extract shows no effect.

[Epidemiological study of high risk human papillomavirus infection in 25 to 54 years old married women in Beijing.].

OBJECTIVE: To investigate high risk human papillomavirus (HR-HPV) prevalence among married women in Beijing and to study the high risk factors. METHODS: During March 2007 to September 2008, a total of 6185 married women sampled from 137 communities in 12 districts were screened by HR-HPV DNA test and cytological test. The interview was carried out with unified questionnaires. The database was set up and twice entered in EpiData 3.0. After checked up, the data were analyzed in SPSS 15.0. RESULTS: (1) The HR-HPV infection rate was 9.89%. The HR-HPV infection rate of the city zone, the suburb and the exurb were 9.34%, 10.51% and 9.51% (P > 0.05). The HR-HPV infection rate of the native and the outlander were 9.53%, 11.30% (P < 0.05). (2) The age distribution of HR-HPV infection was that the rate was around 10% among 25 to 44 age groups, which was the highest (11.21%) in 30 to 34 age group; then the rate was descended as the age raising, the rate of 50 to 54 age group was the lowest (7.78%). (3) Multiple logistic regression showed that the related risk factors of HR-HPV infection mainly included 1000 RMB and above of family income per person per month, possessing more than 1 sexual partner of her husband, outlander and high levels of education. (4) The prevalence of cervical intraepithelial neoplasia (CIN) in HR-HPV positive group was significantly higher than that in HR-HPV negative group (29.76% vs 3.32%, P < 0.01). CONCLUSIONS: (1) The HR-HPV infection rate among aged 25 to 54 years was 9.9% and there was no significant difference in area distribution. (2) The high risk population which should strengthen screening was the married bearing-age women with high level of family income, outlander, high levels of education and her husband possessing more than 1 sexual partner. (3) HR-HPV infection is the main risk factor for CIN and cervical cancer, while does not provide a causal relationship with them. The high risk population should be checked regularly to understand the development of HR-HPV infection and CIN incidence.

The risk factors and prognosis of delayed perihematomal edema in patients with spontaneous intracerebral hemorrhage.

PURPOSE: We hypothesize delayed perihematomal edema (DHE) leads to secondary injury after spontaneous intracerebral hemorrhage (sICH) with a poor prognosis. Hence, we need to investigate the risk factors of DHE and identify whether DHE will predict the poor outcome of sICH. METHODS: We retrospectively recruited 121 patients with sICH admitted to the Department of Neurology from January 2014 to August 2018. After dividing all these patients into DHE group and non-DHE group, we analyzed the potential risk factors and outcome of DHE using a multivariate logistic regression model. RESULTS: We conclude DHE after sICH associates with age, hospitalization time, hematoma shape, blood pressure upon admission, alcohol consumption, blood sodium level, and baseline hematoma volume within 24 hours after symptom onset, among which differences were statistically significant (P < .05). Logistic regression analysis finally identified that age (OR = 0.958, 95% CI = 0.923-0.995) and the baseline hematoma volume (OR = 1.161, 95% CI = 1.089-1.238) were the most significant risk factors for DHE, and moreover, the DHE (OR = 3.062, 95% CI = 1.196-7.839) was also a risk factor for poor prognosis in sICH patients. CONCLUSION: We suggest DHE is a clinical predictor of secondary injury following sICH and poor prognosis. In addition, age and baseline hematoma volume are considered significant high-risk factors for DHE in patients with sICH.

α-Tocopherol protects keratinocytes against ultraviolet A irradiation by suppressing glutathione depletion, lipid peroxidation and reactive oxygen species generation.

This study aimed to investigate whether α-tocopherol is able to protect keratinocytes against ultraviolet A (UVA) radiation by increasing glutathione (γ-glutamylcysteinylglycine; GSH) levels or decreasing lipid peroxidation and reactive oxygen species (ROS) generation. The cell survival fraction was 43.6% when keratinocytes were irradiated with UVA at a dose of 8 J/cm2. α-Tocopherol was added prior to UVA irradiation and the cell viability was assayed. The cell survival fractions were 60.2, 77.1, 89.0, 92.9 and 96.2% when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These results suggested that α-tocopherol is capable of protecting keratinocytes against UVA irradiation. Furthermore, the levels of GSH, lipid peroxidation and ROS were measured. The levels of GSH were 0.354 and 0.600 mmol/g protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 0.364, 0.420, 0.525, 0.540 and 0.545 mmol/g protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of lipid peroxidation were 20.401 or 5.328 µmol/g [malondialdehyde (MDA)/protein] in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 11.685, 6.544, 5.847, 4.390 and 2.164 µmol/g (MDA/protein) when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. The levels of ROS were 3,952.17 or 111.87 1/mg protein in keratinocytes irradiated with UVA (8 J/cm2) and in non-irradiated cells, respectively, whereas they were 742.48, 579.36, 358.16, 285.63 and 199.82 1/mg protein when α-tocopherol was added at concentrations of 2.9, 5.9, 8.8, 11.8 and 14.7 IU/ml, respectively. These findings suggested that α-tocopherol protects keratinocytes against UVA irradiation, possibly through increasing the levels of GSH or decreasing the levels of lipid peroxidation and ROS generation.