[Research progress on the comorbidity between hepatitis B virus infection and noncommunicable diseases].

With the decline in hepatitis B virus (HBV) incidence and the increase in the life expectancy of infected individuals, the population infected with HBV is experiencing rapid aging, leading to an escalating risk of co-morbid chronic noncommunicable diseases (NCDs). This study summarizes research related to the comorbidity between HBV and NCDs, discussing the aging of the HBV-infected population, the mechanisms, prevalence, and management of this comorbidity. This study provides insights into potential directions for future research on the comorbidity between HBV and NCDs and aims to provide a basis for further research and the development of prevention and treatment strategies for the comorbidity of NCDs among HBV-infected individuals in China.

[Efficacy and safety of various doses of hybutimibe monotherapy or in combination with atorvastatin for primary hypercholesterolemia: a multicenter, randomized, double-blind, double-dummy, parallel-controlled phase â ¢ clinical trial].

Objective: To evaluate the efficacy and safety of hybutimibe monotherapy or in combination with atorvastatin in the treatment of primary hypercholesterolemia. Methods: This was a multicenter, randomized, double-blind, double-dummy, parallel-controlled phase Ⅲ clinical trial of patients with untreated primary hypercholesterolemia from 41 centers in China between August 2015 and April 2019. Patients were randomly assigned, at a ratio of 1∶1∶1∶1∶1∶1, to the atorvastatin 10 mg group (group A), hybutimibe 20 mg group (group B), hybutimibe 20 mg plus atorvastatin 10 mg group (group C), hybutimibe 10 mg group (group D), hybutimibe 10 mg plus atorvastatin 10 mg group (group E), and placebo group (group F). After a dietary run-in period for at least 4 weeks, all patients were administered orally once a day according to their groups. The treatment period was 12 weeks after the first dose of the study drug, and efficacy and safety were evaluated at weeks 2, 4, 8, and 12. After the treatment period, patients voluntarily entered the long-term safety evaluation period and continued the assigned treatment (those in group F were randomly assigned to group B or D), with 40 weeks' observation. The primary endpoint was the percent change in low density lipoprotein cholesterol (LDL-C) from baseline at week 12. Secondary endpoints included the percent changes in high density lipoprotein cholesterol (HDL-C), triglyceride (TG), apolipoprotein B (Apo B) at week 12 and changes of the four above-mentioned lipid indicators at weeks 18, 24, 38, and 52. Safety was evaluated during the whole treatment period. Results: Totally, 727 patients were included in the treatment period with a mean age of (55.0±9.3) years old, including 253 males. No statistical differences were observed among the groups in demographics, comorbidities, and baseline blood lipid levels. At week 12, the percent changes in LDL-C were significantly different among groups A to F (all P<0.01). Compared to atorvastatin alone, hybutimibe combined with atorvastatin could further improve LDL-C, TG, and Apo B (all P<0.05). Furthermore, there was no significant difference in percent changes in LDL-C at week 12 between group C and group E (P=0.991 7). During the long-term evaluation period, there were intergroup statistical differences in changes of LDL-C, TG and Apo B at 18, 24, 38, and 52 weeks from baseline among the statins group (group A), hybutimibe group (groups B, D, and F), and combination group (groups C and E) (all P<0.01), with the best effect observed in the combination group. The incidence of adverse events was 64.2% in the statins group, 61.7% in the hybutimibe group, and 71.0% in the combination group during the long-term evaluation period. No treatment-related serious adverse events or adverse events leading to death occurred during the 52-week study period. Conclusions: Hybutimibe combined with atorvastatin showed confirmatory efficacy in patients with untreated primary hypercholesterolemia, which could further enhance the efficacy on the basis of atorvastatin monotherapy, with a good overall safety profile.

[Development of case report form based on clinical data interchange standards consortium on special diseases to promote the ecology construction of real-world data in China].

Real world data (RWD) refers to the data generated in routine clinical practices, daily life, and real work environment and has been widely used in clinical or public health research. Still, issues related to the quality of RWD, such as incompleteness, inconsistency, and inaccuracy, would affect the validity of real-world research. To overcome the challenges due to the lack of standardization of real world source data, case report form based on clinical data interchange standards consortium (CDISC-CRF) on certain diseases was developed to promote the ecology construction of RWD based on the data standards set by the CDISC which has been widely used. Firstly, we described how to apply data standards to make up the gap between RWD and real world evidence. Then, the process was designed to build RWD ecology based on CDISC-CRF, in which the development technology of CDISC-CRF form is mainly introduced. Finally, the application prospect and significance of building real-world data based on disease-specific CDISC-CRF are described. It is believed that the present paper can provide a new idea for promoting the ecology construction of RWD in China.

[Study on mental health status of pregnant women and its influencing factors in the third trimester].

Objective: To understand the mental health status of pregnant women in the third trimester of pregnancy, and explore the influencing factors. Methods: The general demographic information and pregnancy information of 575 pregnant women in the Chinese Pregnant Women Cohort Study were collected. The mental health status of pregnant women in the third trimester was investigated by using Edinburgh Postpartum Depression Scale, Self-Rating Anxiety Scale and University of California at Los Angeles. Multivariate unconditional logistic regression was used to analyze the influencing factors for mental health of pregnant women. Results: In the third trimester of pregnancy, the incidence of depression was 16.52%, the incidence of anxiety was 11.13%, and the incidence of feeling loneliness was 26.26%. Logistic regression analysis showed that compared with those with education level of junior high school below, those with education level of college or bachelor's degree (OR=0.418, 95%CI: 0.184-0.950) and master's degree or above (OR=0.116, 95%CI: 0.027-0.503) were less likely to feel loneliness. Pregnant women with higher annual family income (10 000 RMB yuan) were less likely to suffer from depression (≥20 vs. <10: OR=0.527, 95%CI: 0.279-0.998), anxiety (10-20 vs. <10: OR=0.363, 95%CI: 0.180-0.731; ≥20 vs. <10: OR=0.271, 95%CI: 0.132-0.554) and feeling loneliness (≥20 vs. <10: OR=0.477, 95%CI: 0.276-0.826). Conclusions: The education level and family income have impacts on the mental health status of pregnant women in the third trimester. Medical staff should give targeted psychological advice to pregnant women to improve their mental health status.

[Effects of normobaric hyperoxia intervention on renal ischemia-reperfusion injury in rats].

Objective: To investigate the effects of normobaric hyperoxia intervention on renal ischemia-reperfusion injury in rats and its possible mechanism. Methods: Twenty-one adult male SD rats were enrolled and their right kidneys were excised. After two weeks, they were randomly assigned to 3 groups, with 7 rats in each group, namely sham-operated group (Group S), ischemia-reperfusion group (Group I/R), and normobaric hyperoxia+ischemia-reperfusion group (Group NBHO+I/R). In group S, only the left renal pedicle was isolated, but no ischemic treatment was performed. However, in group I/R and group NBHO+I/R, left renal pedicles were separated and left renal ischemia was induced by noninvasive arterial clamp for 45 min, and after 24 h of reperfusion, rats in group S and group I/R inhaled regular concentration of oxygen (21%), while rats in group NBHO+I/R inhaled high concentration of oxygen (60%), 2 h at each time, once a day for 7 days. On the 7th day after surgery, blood urea nitrogen (BUN) and creatinine (Cr) levels were measured by taking blood from the orbital veins of rats. The content of malondialdehyde (MDA) and superoxide dismutase (SOD) was detected from the left kidney tissues. The mRNA and protein contents of Keap1 and Nrf2 gene in kidney tissues were determined by qPCR and Western Blotting, respectively. Hematoxylin-eosin staining (HE) was employed to observe the pathological changes of kidney tissue. Immunohistochemical staining was used to measure the protein expression of Keap1 and Nrf2 in kidney tissues. Results: Compared with group S, the serum BUN [(10.7±1.7) mmol/L, (8.4±1.0) mmol/L vs (6.1±1.3) mmol/L, both P<0.05] and Cr [(81.0±3.7) µmol/L, (62.9±3.4) µmol/L vs (48.3±2.9) µmol/L, both P<0.05] levels of rats in the group I/R and group NBHO+I/R increased, and the I/R group had the most significant increase. Compared with group S, the MDA content of kidney tissue in the rats of group I/R and NBHO+I/R increased [(10.5±1.0) µmol/L, (8.6±0.8) µmol/L vs (6.5±0.5) µmol/L, both P<0.05], but the MDA content in group NBHO+I/R was lower than that of group I/R (P<0.05). Compared with group S, the SOD content in the kidney tissues of rats in both group I/R and group NBHO+I/R decreased. However, the SOD content of group NBHO+I/R was higher than that of group I/R (P<0.05). Compared with group S, the mRNA and protein contents of Keap1 gene in kidney tissues of group I/R and group NBHO+I/R decreased, and group NBHO+I/R had the most significant decrease (P<0.05). However, compared with group S, mRNA and protein expressions of Nrf2 gene increased in kidney tissues of group I/R and group NBHO+I/R, and NBHO+I/R group had the most significant increase (P<0.05). Postoperative pathological results suggested that compared with group S, the pathological damage of kidney tissues in group I/R and group NBHO+I/R increased, but the degree of damage in group NBHO+I/R was lower than that in group I/R. Conclusion: Normobaric hyperoxia intervention may have protective effects on renal ischemia-reperfusion injury in rats by activating Keap1-Nrf2 signaling pathway.

[Association between parental socioeconomic status and preschoolers' consumption of sugar-sweetened beverages].

Objective: To explore the association between parental socioeconomic status (SES) and preschoolers' consumption of sugar-sweetened beverages (SSB). Methods: In June 2018, all preschoolers from 15 kindergartens were selected from the jurisdiction of Education Commission in Dongcheng District of Beijing by using an equal-proportion stratified cluster sampling method in the study. A self-designed questionnaire was used to investigate the parents of preschoolers to obtain the basic information of preschoolers and parents, the consumption situation of preschoolers' sugar-sweetened beverages and the perception of parents to SSB. A tatol of 3 217 preschoolers were finally included in the analysis. A generalized structural equation model was used to analyze the relationship between preschoolers' consumption of sugar-sweetened beverages and their parents' socioeconomic status and the mediating effect of their cognition of sugar-sweetened beverages. The size of mediating effect was estimated by using deviation correction non-parameter percentile Bootstrap method. Results: The age of 3 217 preschoolers was (4.23±0.67) years, of which 52.6% (n=1 692) were boys, and 77.62% (n=2 497) were SSB consumers. Among the parents of 3 217 preschoolers, fathers and mothers accounted for 24.90% (n=801) and 75.10% (n=2 416), and the M (P25, P75) scores of SES were 66.7 (62.5, 69.5) and 69.5 (64.6, 71.4), respectively. The proportion of parents who took the initiative to learn about their children's consumption of SSB, lacked confidence in restricting preschooler's consumption of SSB and read nutrition labels before purchasing food was 74.08% (n=2 383), 82.90% (n=2 667) and 36.24% (n=1 166), respectively. The generalized structural equation model showed that after adjusting for preschoolers' gender, age, body mass index (BMI) of preschoolers and their parents, preschoolers' consumption of SSB was negatively associated with their parents' SES score [path coefficient (95%CI):-4.69×10-2 (-6.56×10-2,-2.69×10-2) ]. The mediating effect of parents' perception of SSB consumption could explain 48.71% of the total effect [path coefficient (95%CI):-2.28×10-2 (-3.54×10-2, -1.10×10-2)]. Conclusion: The consumption of SSB in preschoolers is negatively associated with their parent's SES, and this relationship is partially mediated by parent's perception of SSB consumption.

[Clinical value of intact parathyroid hormone levels on the first day after total thyroidectomy on prediction for permanent hypoparathyroidism].

Objective: To examine the value serum calcium and intact parathyroid hormone (iPTH) levels measured on the first day after total thyroidectomy on prediction for permanent hypoparathyroidism. Methods: Totally 546 patients with thyroid cancer and benign thyroid lesions who underwent total thyroidectomy at Department of General Surgery, Beijing Chaoyang Hospital, Capital Medical University from February 2008 to December 2018 were analyzed retrospectively. There were 158 males and 388 females aging (50.9±13.2) years (range: 19.0 to 79.2 years). Serum calcium and iPTH levels were collected before surgery, on the first day and 6 months after surgery. Logistic regression was used to analyze the correlation between each data and the occurrence of permanent hypoparathyroidism after surgery.The area under the receiver operating characteristic curve was used to evaluate the predictive power of iPTH for postoperative occurrence of permanent hypoparathyroidism. Results: Among the 546 cases of total thyroidectomy, 22 cases of permanent hypoparathyroidism occurred, with an incidence of 4.0% (22/546). Multivariate analysis showed that iPTH levels on the first day after total thyroidectomy (OR=2.932, 95%CI: 1.129 to 7.616, P=0.027) and serum calcium levels (OR=2.584, 95%CI: 1.017 to 6.567, P=0.046) were independent prognosis factors for postoperative permanent hypoparathyroidism. When the threshold value of iPTH at 24 hours after total thyroidectomy was 5.51 ng/L, the AUC was 0.956 (95%CI: 0.936 to 0.972, P=0.000), sensitivity was 100%, specificity was 85.1%, positive predictive value was 22%, negative predictive value was 100%. When the threshold value of serum calcium at 24 hours after total thyroidectomy was 1.93 mmol/L, the AUC was 0.733 (95%CI: 0.694 to 0.770, P=0.000), sensitivity was 63.6%, specificity was 78.1%, positive predictive value of 10.8% and negative predictive value of 98.1%. Conclusions: Serum iPTH and calcium levels on the first day after total thyroidectomy were related to the occurrence of permanent hypoparathyroidism postoperatively. The predictive value of iPTH level is higher than that of serum calcium level.

[Associations between physical activities and gestational diabetes mellitus in the first trimester of pregnancy].

Objective: To prospectively explore the associations between total and different types of physical activities and gestational diabetes mellitus (GDM) in the first trimester of pregnant women, in Beijing. Methods: Data of 909 participants from the Chinese Pregnant Women Cohort study were extracted and studied. Four types of physical activities, including household/care-giving, occupational, sports/exercise and outdoor ways of transportation (walking/cycling/E-biking) in early pregnancy were studied by using the pregnancy physical activity questionnaire (PPAQ). All the participants were followed up and related data on GDM collected. Logistic regressions were conducted for data analysis on the associations of interests. Results: A total of 206 pregnant women (22.7%) were diagnosed with GDM. After adjusting for age, pre-pregnancy BMI, family history of diabetes and other types of physical activities, results from the logistic regression showed that women with moderate level of household/care-giving activities were with the lower odds on GDM (OR=0.654, 95%CI: 0.436-0.980). Women who had met the guideline of exercise were less likely to have GDM (OR=0.518, 95%CI: 0.287-0.934). There was no significant association appeared between total physical, occupational and outdoor ways of transportation (walking/cycling/E-biking) activities with GDM. Conclusions: Sports/exercise and household/care-giving activities in early pregnancy appeared important on GDM prevention. Pregnant women are encouraged to wisely arranging their sports/exercise activities and engaging in moderate household/care-giving activities in the first trimester of pregnancy, to prevent GDM.

[Effects of changes in physical activities on depressive symptoms during pregnancy: a cohort study].

Objective: To investigate the effect of changes in physical activities on depressive symptoms in the second trimester of pregnancy. Methods: Data from the Chinese Pregnant Women Cohort Study, from July 25, 2017 to November 26, 2018, were used. Women who had effectively completed the survey of physical activity and depressive symptoms in the first and second trimesters of pregnancy, were recruited. Both International Physical Activity Questionnaire-short and Edinburgh Postnatal Depression Scale were used, respectively. Pregnant women who had completed two surveys were included in our study and were divided into two groups according to the depressive symptoms, measured at the baseline. Among the pregnant women without depressive symptoms at the baseline, logistic regression was used to analyze the effects of changes in physical activity on the prevention of depressive symptoms. Effect on the reduction of depressive symptoms was analyzed, using the same method. Results: The prevalence rates of depressive symptoms were 23.83% and 20.57% in the baseline and second trimester, respectively. After adjusting for age, education level, occupation, family annual income and pre-pregnancy BMI, data from the logistic regression showed that women without depressive symptoms and with increased and adequate physical activities in the baseline, were with lower risks to develop depressive symptoms in the second trimester (OR=0.479, 95%CI: 0.335-0.684;OR=0.566, 95%CI: 0.394-0.815). Among women with depressive symptoms in the baseline survey, association between physical activity and depressive symptoms was not statistically significant (P>0.05). Conclusions: Increased and adequate physical activities showed preventive effects on depressive symptoms during pregnancy, but the remission effect was not obvious in women with depressive symptoms. Pregnant women should be encouraged to increase their physical activities while screening programs should also be carried out to reduce the depressive symptoms during pregnancy.

[Factors associated with sleep quality during first and second trimester in Chinese pregnant woman].

Objective: To investigate sleep quality in pregnant women during their first and second trimester and to identify risk factors. Methods: Data was from the Chinese Pregnant Women Cohort Study. A total of 3 618 pregnant women were included, with the exclusion 346 women who had missing information. Sociodemographic, health-related behavior, depression and sleep quality information were collected and analyzed. Logistic regression analysis were used to explore the influencing factors of sleep quality in pregnant women. Results: Among the 3 618 pregnant woman 28.2% had poor sleep quality in their first trimester and 28.7% in the second trimester. 15.2% pregnant women had progressively worse sleep and 13.0% had persistently poor sleep had pregnant women were generally suffered from poor sleep quality, difficulty falling asleep, sleep disorders and daily fatigue. Regular diet (OR=0.75, 95%CI: 0.62-0.92) and work (OR=0.84,95%CI: 0.71-0.99) in the first trimester were protective factors of sleep quality in pregnant women. Age ≥30 year old (OR=1.19, 95%CI: 1.03-1.37), passive smoking (OR=1.18, 95%CI: 1.02-1.36) and depression (OR=2.25, 95%CI: 1.95-2.61) in the first trimester were risk factors. Conclusions: The rate of poor sleep quality are high among Chinese pregnant woman during their first and second trimester. The risk factors of sleep quality are multiple. Regular diet and work, reduction of tobacco exposure, alleviation of depression symptom may help improve sleep quality among pregnant women.

[Risk factors for preterm birth, low birth weight and small for gestational age: a prospective cohort study].

Objective: To study the relationship between exposure factors in early pregnancy and preterm birth (PB), low birth weight (LBW) and small for gestational age (SGA) of neonates. Methods: A total of 3 172 pregnant women who were enrolled in the project of Chinese Pregnant Women Cohort Study-Peking Union Medical College (CPWCS-PUMC) from July 25, 2017 to July 24, 2018 and delivered before December 31, 2018 were selected as subjects in this study. The relationship between exposure factors in early pregnancy and adverse outcomes of neonatal delivery was analyzed by using binary logistic regression analysis. Results: The incidence rates of PB, LBW and SGA were 4.76%, 3.53% and 5.74%, respectively. In terms of PB, the analysis results showed that the gestational weight gain (GWG) and living in northern China were protective factors, while premature rupture of membranes, gestational hypertension, dental examination or treatment within 1-3 years and family with 3-4 members were risk factors. In the respect of LBW, GWG and daily consumption of milk and dairy products were the protective factors, while premature rupture of membranes, gestational hypertension, sedentary working time more than 6 hours, dental examination or treatment within 1-3 years and passive smoking were risk factors. For SGA, baby girl, passive smoking, peanut oil consumption and unsalted taste were risk factors, while folic acid supplementation was protective factor. Conclusion: The risk factors for PB, LBW and SGA were multifactorial, and relevant specific measures should be taken to reduce the occurrence of adverse neonatal outcomes.

[Analysis of the arthropathies on no-bleeding history joints in pre-school age severe hemophilia A children].

Objective: To detect the arthropathies on no bleeding history joints in pre-school hemophilia A children in order to provide evidence for further prevention and control of joint disease in children with hemophilia A. Methods: This study was a cross-sectional study based on China Hemophilia Individualized prophylaxis study (CHIPS). The basic data of outpatients with hemophilia in Beijing Children's Hospital and Chengdu Women's and Children's Central Hospital between August 2016 and June 2017 were collected and a three-month follow-up was conducted. The target joints (six joints of bilateral elbows, knees and ankles) of thirty-four children aged 1-7 years old with severe hemophilia A were examined by ultrasound, X-ray and joint function examination (4-7 years old, hemophilia joint health score (HJHS)). To find out whether there are arthropathies in patient's joints with no bleeding history and analyze the relevant factors by chi-square test, rank sum test and other statistical methods. Results: There were 32 analyzable cases with 112 no-bleeding history target joints, 42.9% (48/112) were elbow joints. Arthropathies were revealed in 34.8% (39/112) of them by joint structural and functional examination and 46.2% (18/39) were ankles (χ(2)=8.379, P=0.015) . Ultrasound showed abnormalities in 18.3% (20/109) joints, X-ray showed abnormalities in 3.8% (3/79) joints and HJHS showed abnormalities in 25.3% (20/79) joints. There was no correlation between ultrasound and HJHS (r=0.015, P=0.895), no correlation was found between X-ray and HJHS (r=-0.101, P=0.390) either, which suggested that joint structural and functional examination could not replace each other. The related risk factors of arthropathies in this group were >4.91 years old (OR=3.917, 95%CI:1.610-9.528) and combining with target joint (OR=3.530, 95%CI:1.316-9.465). Conclusions: Detecting the joint structure and function on no bleeding history joints in pre-school hemophilia A children could reveal the arthropathies and majority of them were ankle arthropathies. Joint structural and functional examinations could not replace each other. For patients more than 5 years old and those with target joints, the joints with no complaint of bleeding should be examined regularly to reveal the arthropathies in time.

[The application of Hoffmann method in the establishment of children's reference intervals of erythrocyte count].

OBJECTIVE: To verify the reliability and feasibility of Hoffmann method in establishing pediatric reference intervals (RI) of erythrocyte count. METHODS: Three hundreds and ninty-two thousands of hospital-based data for erythrocyte count of children aged in 1 to 17, measured by the Sysmex Xs-800i, was collected from Beijing Children's Hospital during January to December 2014. Outliers were removed using the Dixon method, then Hoffmann method was conducted to establish the gender and age stratified pediatric RIs of erythrocyte count. The erythrocyte count of 2 217 healthy children, recruited from Beijing Children's Hospital and Liaocheng Children's Hospital in Shandong province, was conducted as normal reference to verify the reliability of Hoffmann method in establishing RIs and to compare with existing RIs. RESULTS: In 4 subgroups as following, male aging 1 to 12 years, male aging 13 to 17 years, female aging 1 to 12 years, female aging 13 to 17 years, the RIs of erythrocyte count established using Hoffmann method were (4.1-5.4)×10(12)/L, (4.4-5.7)×10(12)/L, (4.0-5.3)×10(12)/L, (4.0-5.3)×10(12)/L, respectively. The verification results in 2 217 healthy children showed that the proportions of out of range in four subgroups were 6.17%, 8.81%, 6.22%, 7.78%, respectively. CONCLUSION: Hoffmann method produce reliable RIs according with the actual situation in healthy children, which is also convenient and is worth popularizing in clinical practice.

[The inflammatory pattern and the characteristics of mucous remodeling in different immune type of nasal polyps].

Objective:The aim of this study is to study the different immune-type of polyps and valuate mucosal inflammatory pattern and remodeling features between in IL-5 positive vs.IL-17 positive nasal polyps.Method:Nasal polyp or nasal turbinate tissue was obtained from 88 CRSwNP patients during endonasal sinus surgery or 18 non-atopic control subjects during septoplasty,respectively.Assessment of pro-inflammatory cytokines and mediators by ELISA.Additionally,the distribution of IL-5 positive or IL-17 positive cells and inflammatory cells(eosinophil,neutrophil,etc.) were examined using immunohistochemistry(IHC).Result:Overall more than half amount of polyp tissue did not express any TH cells key cytokine.However there are 21% polyp present IL-5 positive and 16% of IL-17 positive as well as 9% of IFN-γ positives.Amount of them that IL-17 positive polyps by synthesis of mediators promoting neutrophilic inflammation[myeloperoxidase(MPO),IL-1ß,IL-6 and IL-8)] and staining infiltration of MPO positive with IL-17 positive cells,whereas IL-5 positive nasal polyps were characterized by synthesis of mediators promoting eosinophilic inflammation(IL-5,ECP,TIgE,and SAE-IgE) and infiltrating of eosinophils and IL-5 positive cells.Meanwhile TGF-ß1 and MMP7 protein levels enhanced in IL-17 positive polyps and decreased in IL-5 positive than control.Conclusion:Nasal polyps presenting as different immune types and there were characterized with different inflammatory and remodeling patterns.Amount of them that IL-17 positive polyps presenting as a neutrophilic inflammation with remodeling biased.Whereas IL-5 positive nasal polyps were characterized by eosinophilic inflammation and absence of remodeling tendency.

Caspase-dependent deubiquitination of monoubiquitinated nucleosomal histone H2A induced by diverse apoptogenic stimuli.

Enzymatic deubiquitination of mono-ubiquitinated nucleosomal histone H2A (uH2A) and H2B (uH2B) is closely associated with mitotic chromatin condensation, although the function of this histone modification in cell division remains ambiguous. Here we show that rapid and extensive deubiquitination of nucleosomal uH2A occurs in Jurkat cells undergoing apoptosis initiated by anti-Fas activating antibody, staurosporine, etoposide, doxorubicin and the proteasome inhibitor, N-acetyl-leucyl-leucyl-norlucinal. These diverse apoptosis inducers also promoted the accumulation of slowly migrating, high molecular weight ubiquitinated proteins and depleted the cellular pool of unconjugated ubiquitin. In apoptotic cells, ubiquitin was cleaved from uH2A subsequent to the appearance of plasma membrane blebbing, and deubiquitination of uH2A closely coincided with the onset of nuclear pyknosis and chromatin condensation. Nucleosomal uH2A deubiquitination, poly (ADP-ribose)polymerase (PARP) cleavage and chromatin condensation were prevented in cells challenged with apoptosis inducers by pretreatment with the pan-caspase inhibitor, zVAD-fmk, or by over-expressing anti-apoptotic Bcl-xL protein. These results implicate a connection between caspase cascade activation and nucleosomal uH2A deubiquitination. Transient transfection of 293 cells with the gene encoding Ubp-M, a human deubiquitinating enzyme, promoted uH2A deubiquitination, while an inactive mutated Ubp-M enzyme did not. However, Ubp-M-promoted deubiquitination of uH2A was insufficient to initiate apoptosis in these cells. We conclude that uH2A deubiquitination is a down-stream consequence of procaspase activation and that unscheduled cleavage of ubiquitin from uH2A is a consistent feature of the execution phase of apoptosis rather than a determining or initiating apoptogenic event. Nucleosomal uH2A deubiquitination may function as a cellular sensor of stress in situations like apoptosis through which cells attempt to preserve genomic integrity.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations.

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 microM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

A primitive ATP receptor from the little skate Raja erinacea.

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.

A mutant deubiquitinating enzyme (Ubp-M) associates with mitotic chromosomes and blocks cell division.

A new ubiquitin-processing protease (Ubp-M) has been identified in mammalian cells that is phosphorylated at the onset of mitosis and dephosphorylated during the metaphase/anaphase transition. The carboxyl-terminal domain of this 823-aa protein can be phosphorylated in vitro with either extracts of mitotic cells or purified cdc-2/cyclin B complexes. Recombinant Ubp-M is able to deubiquitinate histone H2A in vitro, and the phosphorylated form is also enzymatically active. Wild-type Ubp-M, transiently expressed as green fluorescent protein-fusion proteins, localizes in the cytoplasm of cultured cells, but mutant forms, lacking an active-site cysteine, associate closely with mitotic chromosomes during all stages of cell division and remain within the nucleus during the postmitotic period. Cells transfected with plasmids containing mutant Ubp-M genes stop dividing and eventually undergo apoptosis. Ubp-M may deubiquitinate one or more critical proteins that are involved in the condensation of mitotic chromosomes, possibly acting selectively on histones H2A and H2B, the major ubiquitinated proteins of chromatin.

Engineered bacterial Fc receptors.

Five new-type Fc receptor molecules were constructed based on streptococcal protein G (SpG) and staphylococcal protein A (SpA). These protein molecules contain one to six Fc binding domains to immunoglobulins which are structurally different from native SpG or SpA. Their expression levels reached 17-30% of the total bacterial proteins after heat induction in E. coli. Immunodiffusion and ELISA results showed that the engineered protein TG (184 amino acid residues) composed of three SpG C3 domain could bind more broadly and efficiently than the native SpG to the IgGs of human, goat, rabbit, etc., and its optimal pH for binding became wider (pH5-8) compared with the SpG (pH5); and the protein TGA (357AA), fused by protein TG and the A, B, C domains of SpA, displayed both the binding pattern of SpG and SpA.

Structure analysis of streptococcal protein G Fc binding domain.

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein G nucleotide sequences, and results in an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA. The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm. It shows a typical alpha-helix region in this domain. By breaking this alpha-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG. The hydropathicity of this domain was also analyzed and compared with that of protein A relevant domain. Some similarity was found. These results suggest that the binding mechanism of protein G to the IgG Fc part depends on hydrophobic action which comes from the alpha-helix in protein G molecule, just as protein A binding to IgG Fc part.