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Rationale: The degree to which chronic respiratory health effects caused by exposures to cotton dust and endotoxin is reversible after cessation of textile work is unknown.Objectives: To investigate changes in lung function and respiratory symptoms after cessation of textile work and to determine whether past exposure to cotton dust and endotoxin or smoking history modify the associations.Methods: We performed a prospective cohort study consisting of 447 cotton textile workers exposed to cotton dust and 472 unexposed silk textile workers, with a 25-year follow-up. Spirometry testing and respiratory questionnaires were conducted at 5-year intervals. Generalized estimated equations were used to model the average 5-year change in FEV(1) and odds ratios of respiratory symptom prevalence.Measurements and Main Results: Years since cessation of textile work was positively associated with 11.3 ml/yr and 5.6 ml/yr gains in 5-year FEV(1) change for cotton and silk workers, respectively. Among male cotton workers, smokers gained more FEV(1) per year after cessation of exposure than did nonsmokers, and the risk of symptoms of chronic bronchitis and byssinosis was larger for smoking than for nonsmoking male cotton workers.Conclusions: Cessation of textile work was significantly associated with improvement in lung function and respiratory symptoms. The positive effect of work cessation was greater for cotton workers than for silk workers. For cotton workers, the improvement in lung function loss after cessation of textile work was greater among smokers, but no differences were observed for silk workers. [ABSTRACT FROM AUTHOR]

Rationale: Acute airway response, measured as cross-shift change in FEV(1), to cotton dust may lead to subsequent chronic loss of lung function in exposed workers.Objectives: To explore the association between the magnitude and frequency of cross-shift change and chronic loss of FEV(1).Methods: Four hundred eight cotton workers and 417 silk workers from Shanghai textile mills were observed prospectively for 20 years, with cross-shift measurements at baseline and follow-up surveys at approximate 5-year intervals. To account for repeated measures of 5-year change, generalized estimating equations were used to estimate the relationship between the magnitude of cross-shift change in FEV(1) (DeltaFEV(1)) and subsequent 5-year annualized change. Linear regression models were used to examine the association between the number of drops in cross-shift FEV(1) (DeltaFEV(1) < 0) and annualized change over the entire study period.Measurements and Main Results: Exposure to cotton dust was associated with a 10 ml/year decrement in 5-year annualized FEV(1) decline. In addition, every 10 ml in DeltaFEV(1) drop was associated with an additional 1.5 ml/year loss in annualized FEV(1) decline. The association between the frequency of drops and annualized decline was stronger for cotton workers than for silk workers over the entire study period.Conclusions: Cotton workers had larger and more frequent drops, as well as excessive chronic declines in FEV(1), than did silk workers. The magnitude and frequency of cross-shift drops were associated with chronic loss in FEV(1) over the entire 20-year period examined. [ABSTRACT FROM AUTHOR]

Background: Bronchiectasis is a highly heterogeneous chronic airway disease with marked geographic and ethnic variations. Most influential cohort studies to date have been performed in Europe and USA, which serve as the examples for developing a cohort study in China where there is a high burden of bronchiectasis. The Establishment of China Bronchiectasis Registry and Research Collaboration (BE-China) is designed to: (1) describe the clinical characteristics and natural history of bronchiectasis in China and identify the differences of bronchiectasis between the western countries and China; (2) identify the risk factors associated with disease progression in Chinese population; (3) elucidate the phenotype and endotype of bronchiectasis by integrating the genome, microbiome, proteome, and transcriptome with detailed clinical data; (4) facilitate large randomized controlled trials in China., Methods: The BE-China is an ongoing prospective, longitudinal, multi-center, observational cohort study aiming to recruit a minimum of 10,000 patients, which was initiated in January 2020 in China. Comprehensive data, including medical history, aetiological testing, lung function, microbiological profiles, radiological scores, comorbidities, mental status, and quality of life (QoL), will be collected at baseline. Patients will be followed up annually for up to 10 years to record longitudinal data on outcomes, treatment patterns and QoL. Biospecimens, if possible, will be collected and stored at - 80 °C for further research. Up to October 2021, the BE-China has enrolled 3758 patients, and collected 666 blood samples and 196 sputum samples from 91 medical centers. The study protocol has been approved by the Shanghai Pulmonary Hospital ethics committee, and all collaborating centers have received approvals from their local ethics committee. All patients will be required to provide written informed consent to their participation., Conclusions: Findings of the BE-China will be crucial to reveal the clinical characteristics and natural history of bronchiectasis and facilitate evidence-based clinical practice in China. Trial registration Registration Number in ClinicalTrials.gov: NCT03643653., (© 2022. The Author(s).)

Vancomycin remains the mainstay of treatment for methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. This study assessed risk factors for vancomycin failure in 63 patients with MRSA pneumonia through detailed clinical, microbiological, pharmacokinetic/pharmacodynamic, and genetic analyses of prospective multicenter studies conducted from February 2012 to July 2018. Therapeutic drug monitoring was performed during vancomycin treatment, and the 24-h area under the curve (AUC 0-24 ) was calculated. All baseline strains were collected for MIC determination, heterogeneous vancomycin-intermediate S. aureus (hVISA) screening, and biofilm determination. Whole-genome sequencing was performed on the isolates to analyze their molecular typing and virulence and adhesion genes. Clinical signs and symptoms improved in 44 patients (44/63, 69.8%), with vancomycin daily dose ( P =  0.045), peak concentration ( P =  0.020), and sdrC ( P =  0.047) being significant factors. Isolates were eradicated in 51 patients (51/63, 81.0%), with vancomycin daily dose ( P =  0.009), cardiovascular disease ( P =  0.043), sequence type 5 (ST5; P = 0.017), tst ( P =  0.050), and sec gene ( P =  0.044) associated with bacteriological failure. Although the AUC 0-24 /MIC was higher in the groups with bacterial eradication, the difference was not statistically significant ( P =  0.108). Multivariate analysis showed that no variables were associated with clinical efficacy; ST5 was a risk factor for bacterial persistence (adjusted odds ratio, 4.449; 95% confidence interval, 1.103 to 17.943; P =  0.036). ST5 strains had higher frequencies of the hVISA phenotype, biofilm expression, and presence of some adhesion and virulence genes such as fnbB , tst , and sec than non-ST5 strains. Our study suggests that ST5 is a possible predictor of bacterial persistence in MRSA pneumonia treated with vancomycin. IMPORTANCE Few studies have simultaneously examined the influence of clinical characteristics of patients with pneumonia, the vancomycin pharmacokinetic/pharmacodynamic (PK/PD) index, and the phenotypic and genetic characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains. We assessed risk factors for vancomycin failure in patients with MRSA pneumonia by analyzing these influences in a prospective multicenter study. Sequence type 5 (ST5) was a possible predictor of bacterial persistence in adult patients with MRSA pneumonia (adjusted odds ratio, 4.449). We found that this may be related to ST5 strains having higher levels of vancomycin heterogeneous resistance, biofilms, and the presence of adhesion and virulence genes such as fnbB , tst , and sec .

Background: Patients with chronic obstructive pulmonary disease (COPD) are more susceptible to Aspergillus colonization or infection. Several studies have demonstrated that invasive pulmonary Aspergillosis (IPA) and Aspergillus hypersensitivity (AH) have a detrimental effect on COPD. However, it remains to be clarified whether Aspergillus colonization is associated with acute exacerbation of COPD (AECOPD). This study aimed to explore the impact of Aspergillus colonization in the lower respiratory tract on AECOPD. Method: Patients with Aspergillus colonization were identified from a retrospective cohort of hospitalized AECOPD from 2011 to 2016 in eight centers in Shanghai, China. The demographic information, conditions of the stable stage, clinical characteristics during hospitalization, and 1-year follow-up information after discharge were collected and compared to participants without fungi colonization. Result: Twenty-six hospitalized AECOPD patients with Aspergillus colonization and 72 controls were included in the final analysis after excluding patients with other fungi isolation and matching. The rates of recurrence of acute exacerbation within 90 days and 180 days after discharge in the patients with Aspergillus colonization were both significantly higher than that in the fungi negative patients (90 days: 19.2 vs. 4.2%, p = 0.029; 180 days: 23.1 vs. 4.2%, p = 0.010), and the all-cause mortality within 1 year was also higher (11.5 vs. 0.0%, p = 0.017). Multivariate logistic regression analysis showed that Aspergillus colonization was an independent risk factor for the recurrence of acute exacerbation within 90 days and 180 days (90 days: OR = 8.661, 95% CI: 1.496-50.159, p = 0.016; 180 days: OR =10.723, 95% CI: 1.936-59.394, p = 0.007). Conclusion: Aspergillus colonization may predict poor prognosis of AECOPD while leading to an increased risk of recurrent AECOPD in a short period., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Zuo, Cheng, Huang, Bao, Jin, Gao, Tu, Hu, Hang, Wang, Zhang and Zhang.)

Objective: To investigate the epidemiology, clinical features, treatment and outcome of Noninvasive ventilation (NIV)-treated acute exacerbation of chronic obstructive pulmonary disease (AECOPD) patients in secondary hospitals of Shanghai., Method: Relying on Shanghai alliances for respiratory diseases, a retrospective observational study was performed in 34 secondary hospitals of Shanghai. The AECOPD patients treated with NIV and admitted to the respiratory department or respiratory intensive care unit were recruited between December 1, 2016, and November 30, 2017., Results: There were 555 patients finally recruited in this study. The age was 75.8 ± 9.6 years old and 380 patients (68.5%) were male. 70.5% of all patients had respiratory acidosis (pH <7.35). 55.3% of all patients received nebulised bronchodilator and 77.7% were treated with systemic or inhaled corticosteroids during hospitalisation. 525 patients (94.6%) recovered successfully and the mortality was 3.2%. The hospitalisation was 15.3 ± 6.7 days and hospital expenses were 22 911 ± 13 595 RMB. Inadequate and nonstandard drug treatments were the most important problems during management., Conclusion: The NIV can be successfully used for AECOP patients in local hospitals of Shanghai, but accompanied by high costs and long hospital stays. However, the treatments for exacerbation and stable COPD patients are still insufficient., (© 2021 John Wiley & Sons Ltd.)

Background: It has been noted that there is an increase in the incidence of acute cardiovascular events (CVEs) in patients with chronic obstructive pulmonary disease (COPD) during an acute exacerbation (AE), thereby causing increased inpatient mortality. Thus, we have tried to identify predictors of acute CVEs in patients with AECOPD via a nested case-control study., Methods: A total of 496 cases hospitalized for AECOPD were included in this study, and followed-up for up to 6 months after discharge. Acute CVEs in the AE period were defined as a new or worsening acute coronary syndrome (ACS), arrhythmia, or left ventricular disfunction (LVD). Predictors of CVEs were selected from several variables, including baseline characteristics and treatments in the stable period as well as symptoms, laboratory tests, complications and treatments in the AE period., Results: Thirty cases (6.05%) had acute CVEs, namely 2 had ACS, 13 had LVD and 19 experienced some form of arrhythmia. Four deaths were observed in the CVE group, with significantly increased death risk compared with the non-CVE group (P = 0.001, OR = 5.81). Moreover, patients who had CVEs were more prone to have re-exacerbation within 3 months. Multivariate analysis showed that previous LVD history (P = 0.004, OR = 5.06), 20% increase in heart rate (HR) (P = 0.003, OR = 10.19), electrolyte disturbance (P = 0.01, OR = 4.24) and diuretics (P = 0.002, OR = 6.37) were independent predictors of CVEs. In addition, usage of theophylline, fluoroquinolone and inhaled beta agonists in the AE period were not statistically associated with acute CVEs., Conclusions: Our preliminary study indicates that patients hospitalized for AECOPD with previous LVD history or increased HR need close observation and diuretics should be cautiously used with regular electrolyte monitoring. These findings need to be confirmed in a large cohort.

Objective: To explore impact of Candida on the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) outcome. Methods: A retrospective, multi-center, case-control study was performed. Patients hospitalized for AECOPD in 25 centers during Jan 2011-Dec 2016 were enrolled. Data were collected, including demographic information, conditions during the stable phase of COPD, clinical characteristics of AECOPD, and follow-up information within 1 year after discharge. Univariate analysis and binary logistic regression were applied, and p < 0.05 was regarded as significant. Results: Totally 1,103 patients were analyzed, with 644 lower respiratory airway (LTR) Candida positive cases and 459 Candida negative controls. Long-term prognosis was significantly different between Candida positive and negative group, including the recurrent AECOPD within 180 days (75.5 vs. 6.6%, p < 0.001) and mortality within 1 year (6.9 vs. 0.4%, p < 0.001). Univariate logistic analysis showed that LTR Candida isolation was related to higher recurrence rate of AECOPD within 180 days and mortality within 1 year. Binary logistic regression analysis demonstrated that LTR Candida isolation was independently associated with recurrence of AECOPD within 180 days. Conclusions: LTR Candida isolation was associated with worse long-term prognosis of AECOPD and independently related to higher risks of recurrent AECOPD within 180 days., (Copyright © 2020 Zuo, Wang, Chen, Liu, Zhang, Jin, Hang, Li, Bao, Jie, Wang, Gao, Sun, Xu, Zhang and Qu.)

Background: Despite the release of a national guideline in 2016, the actual practices with respect to adult community-acquired pneumonia (CAP) remain unknown in China. We aimed to investigate CAP patient management practices in Shanghai to identify potential problems and provide evidence for policy making., Methods: A short-period, 5-day prospective cross-sectional study was performed with sampled pulmonologists from 36 hospitals, encompassing all the administrative districts of Shanghai, during January 8-12, 2018. The medical information was recorded and analyzed for the patients with the diagnosis of CAP who were cared for by 46 pulmonologists during the study period., Results: Overall, 435 patients were included in the final analysis, and 94.3% had a low risk of death in terms of CRB-65 criteria (C: disturbance of consciousness, R: respiratory rate, B: blood pressure, 65: age). When diagnosed with CAP, 70.1% of patients were not evaluated using the CURB-65 score (CRB-65 + U: urea nitrogen), but most patients (95.4%) were evaluated using CRB-65. Time to achieve clinical stability was longer in patients with hypoxemia than in those without hypoxemia (8.42±6.36 vs. 5.53±4.12 days, P=0.004). Overall, 84.4% of patients with a CRB-65 score of 0 were administered antibiotics intravenously, and 19.4% were still hospitalized after excluding hypoxemia and comorbidities. The average duration of antibiotic treatment was 10.4±4.9 days. Overall, 72.6% of patients received antibiotics covering atypical pathogens whose time to clinical stability was significantly shortened compared with those without coverage, but the antibiotic duration was similar and not correspondingly shortened., Conclusions: CRB-65 seems to be more practical than CURB-65 for the initial evaluation of CAP in the context of local practice, and oxygenation assessment should be included in the evaluation of severity. Overtreatment may be relatively common in patients at low risk of death, including unreasonable hospitalization, intravenous administration, and antibiotic duration., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/jtd.2020.03.02). The authors have no conflicts of interest to declare., (2020 Journal of Thoracic Disease. All rights reserved.)

Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active β-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human β-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a β-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40 low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.

Acute exacerbation (AE) is the main cause of increased disability and mortality for patients with Chronic Obstructive Pulmonary Disease (COPD). Short-term re-exacerbation after discharge is common for in-hospital patients with AECOPD. Thus, we aimed to design a scoring system to effectively predict the 30-day re-exacerbation using simple and easily accessible variables. We retrospectively enrolled 686 cases hospitalized for AECOPD in two Chinese hospitals from 2005 to 2017. A variety of parameters were collected like demographics, clinical manifestations and treatments in stable and AE period. The optimal subset of covariates in the multivariate logistic analysis was identified by the smallest Akaike Information Criterion (AIC) and was further used to develop a practical and reliable nomogram to predict the 30-day re-exacerbation. The efficacy of the nomogram was internally validated by concordance index (C-index) and a calibration plot. The incidence of 30-day re-exacerbation was 15.8%. Based on the smallest AIC, eight easily-accessible parameters were included in the nomogram, including sex, COPD assessment test (CAT) scores, AE with respiratory failure in the previous year, new purulent sputum, new cardiovascular events, combined antibiotic therapy, theophylline therapy for AE and ICU admission. Our nomogram revealed good discriminative ability with the C-index of 0.702. The calibration curve showed good agreement between nomogram-predicted probability and actual observation. Incorporating eight common variables, a nomogram for 30-day re-exacerbation after discharge with high predictive performance was constructed for patients with AECOPD, which was helpful in predicting individualized risk of re-exacerbation and offering individualized post-discharge support.

Background: Although occupational exposures contribute to a significant proportion of obstructive lung disease, the phenotype of obstructive lung disease associated with work-related organic dust exposure independent of smoking remains poorly defined., Objective: We identified the relative contributions of smoking and occupational endotoxin exposure to parenchymal and airway remodeling as defined by quantitative computed tomography (CT)., Methods: The Shanghai Textile Worker Study is a longitudinal study of endotoxin-exposed cotton workers and endotoxin-unexposed silk workers that was initiated in 1981. Spirometry, occupational endotoxin exposure, and smoking habits were assessed at 5-year intervals. High-resolution computed tomography (CT) was performed in 464 retired workers in 2011, along with quantitative lung densitometric and airway analysis., Results: Significant differences in all CT measures were noted across exposure groups. Occupational endotoxin exposure was associated with a decrease (-1.3%) in percent emphysema (LAAI-950), a 3.3-Hounsfield unit increase in 15th percentile density, an 18.1-g increase in lung mass, and a 2.3% increase in wall area percent. Current but not former smoking was associated with a similar CT phenotype. Changes in LAAI-950 were highly correlated with 15th percentile density (correlation -1.0). Lung mass was the only measure associated with forced expiratory volume in 1 sec (FEV1) decline, with each 10-g increase in lung mass associated with an additional loss (-6.1 mL) of FEV1 (p = 0.001) between 1981 and 2011., Conclusions: There are many similarities between the effects of occupational endotoxin exposure and those of tobacco smoke exposure on lung parenchyma and airway remodeling. The effects of occupational endotoxin exposure appear to persist even after the cessation of exposure. LAAI-950 may not be a reliable indicator of emphysema in subjects without spirometric impairment. Lung mass is a CT-based biomarker of accelerated lung function decline., Citation: Lai PS, Hang J, Zhang F, Sun J, Zheng BY, Su L, Washko GR, Christiani DC. 2016. Imaging phenotype of occupational endotoxin-related lung function decline. Environ Health Perspect 124:1436-1442; http://dx.doi.org/10.1289/EHP195., Competing Interests: The authors declare they have no actual or potential competing financial interests.

Exposure to air pollution can be particularly high during commuting and may depend on the mode of transportation. We investigated the impact of commuting mode on pulmonary function in Shanghai, China.The Shanghai Putuo Study is a cross-sectional, population-based study. Our primary outcomes were forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) % predicted, and the secondary outcome was spirometric airflow obstruction. We tested the association between mode of transportation and these outcomes after adjusting for confounders.The study population consisted of 20 102 subjects. After adjusting for confounders, the change (95% CI) in FEV1 was -2.15% pred (-2.88- -1.42% pred) among pedestrians, -1.32% pred (-2.05- -0.59% pred) among those taking buses without air conditioning, -1.33% pred (-2.05- -0.61% pred) among those taking buses with air conditioning and -2.83% pred (-5.56- -0.10% pred) among those using underground railways, as compared to cyclists (the reference group). The effects of mode on FVC % predicted were in the same direction. Private car use had a significant protective effect on FVC % predicted and the risk of airflow obstruction (defined by Global Initiative for Chronic Obstructive Lung Disease but not by lower limit of normal criteria).Mode of transportation is associated with differences in lung function, which may reflect pollution levels in different transportation microenvironments., (Copyright ©ERS 2016.)

Background: An inverse association between socioeconomic status and pulmonary function has emerged in many studies. However, the mediating factors in this relationship are poorly understood, and might be expected to differ between countries. We sought to investigate the relationship between socioeconomic status and lung function in China, a rapidly industrializing nation with unique environmental challenges, and to identify potentially-modifiable environmental mediators., Methods: We used data from the Shanghai Putuo Study, a cross-sectional study performed in Shanghai, China. Participants completed a questionnaire and spirometry. The primary exposure was socioeconomic status, determined by education level. The primary outcomes were FEV1 and FVC percent predicted. Multiple linear regressions were used to test this association, and the percent explained by behavioral, environmental, occupational, and dietary variables was determined by adding these variables to a base model., Results: The study population consisted of a total of 22,878 study subjects that were 53.3 % female and had a mean age of 48. In the final multivariate analysis, the effect estimates for FEV1 and FVC percent predicted for low socioeconomic status (compared to high) were statistically significant at a p-value of <0.01. Smoking, biomass exposure, mode of transportation to work, a diet low in fruits or vegetables, and occupational category partially attenuated the relationship between SES and lung function. In a fully-adjusted age-stratified analysis, the socioeconomic disparity in lung function widened with increasing age., Conclusions: We found cross-sectional evidence of socioeconomic disparities in pulmonary function in Shanghai. These differences increased with age and were partially explained by potentially modifiable exposures.

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.

Objectives: The purpose of this study is to determine the trajectory of lung function change after exposure cessation to occupational organic dust exposure, and to identify factors that modify improvement., Methods: The Shanghai Textile Worker Study is a longitudinal study of 447 cotton workers exposed to endotoxin-containing dust and 472 silk workers exposed to non-endotoxin-containing dust. Spirometry was performed at 5-year intervals. Air sampling was performed to estimate individual cumulative exposures. The effect of work cessation on forced expiratory volume in 1 s (FEV1) was modelled using generalised additive mixed effects models to identify the trajectory of FEV1 recovery. Linear mixed effects models incorporating interaction terms were used to identify modifiers of FEV1 recovery. Loss to follow-up was accounted for with inverse probability of censoring weights., Results: 74.2% of the original cohort still alive participated in 2011. Generalised additive mixed models identified a non-linear improvement in FEV1 for all workers after exposure cessation, with no plateau noted 25 years after retirement. Linear mixed effects models incorporating interaction terms identified prior endotoxin exposure (p=0.01) and male gender (p=0.002) as risk factors for impaired FEV1 improvement after exposure cessation. After adjusting for gender, smoking delayed the onset of FEV1 gain but did not affect the overall magnitude of change., Conclusions: Lung function improvement after cessation of exposure to organic dust is sustained. Endotoxin exposure and male gender are risk factors for less FEV1 improvement., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: Airborne endotoxin exposure has adverse and protective health effects. Studies show men have augmented acute inflammatory responses to endotoxin. In this longitudinal cohort study we investigated the effect of long-term exposure to endotoxin in cotton dust on health, and determined whether these effects differ by gender., Methods: In the Shanghai Textile Worker Study, 447 cotton and 472 control silk textile workers were followed from 1981 to 2011 with repeated measures of occupational endotoxin exposure, spirometry and health questionnaires. Impaired lung function was defined as a decline in forced expiratory volume in one second to less than the 5th centile of population predicted. Death was ascertained by death registries. We used Cox proportional hazards models to assess the effect of endotoxin exposure on the time to development of impaired lung function and death., Results: 128 deaths and 164 diagnoses of impaired lung function were ascertained between 1981 and 2011. HRs for the composite end point of impaired lung function or death was 1.47 (95% CI 1.09 to 1.97) for cotton vs silk workers and 1.04 (95% CI 1.01 to 1.07) per 10 000 endotoxin units (EU)/m(3)-years increase in exposure. HRs for all-cause mortality was 1.36 (95% CI 0.93 to 1.99) for cotton vs silk workers and 1.04 (95% CI 0.99 to 1.08) per 10 000 EU/m(3)-years. The risk associated with occupational endotoxin exposure was elevated only in men., Conclusions: Occupational endotoxin exposure is associated with an increase in the risk of impaired lung function and all-cause mortality in men.

Background: Although occupational exposure to cotton dust and endotoxin is associated with adverse respiratory health, associations with cancer are unclear. We investigated cancer mortality in relation to cotton dust and endotoxin exposure in the Shanghai textile workers cohort., Methods: We followed 444 cotton textile and a reference group of 467 unexposed silk workers for 30 years (26 777 person-years). HRs for all cancers combined (with and without lung cancer) and gastrointestinal cancer were estimated in Cox regression models as functions of cotton textile work and categories of cumulative exposure (low, medium, high), after adjustment for covariates including pack-years smoked. Different lag years accounted for disease latency., Results: Risks of mortality from gastrointestinal cancers and all cancers combined, with the exclusion of lung cancer, were increased in cotton workers relative to silk workers. When stratified by category of cumulative cotton exposure, in general, risks were greatest for 20-year lagged medium exposure (all cancers HR=2.7 (95% CI 1.4 to 5.2); cancer excluding lung cancer HR=3.4 (1.7-7.0); gastrointestinal cancer HR=4.1 (1.8-9.7)). With the exclusion of lung cancer, risks of cancer were more pronounced. When stratified by category of cumulative endotoxin exposure, consistent associations were not observed for all cancers combined. However, excluding lung cancer, medium endotoxin exposure was associated with all cancers and gastrointestinal cancer in almost all lag models., Conclusions: Cotton dust may be associated with cancer mortality, especially gastrointestinal cancer, and endotoxin may play a causative role. Findings also indirectly support a protective effect of endotoxin on lung cancer.

Objective: To understand allergic diseases related factors in Changzheng Town, Putuo District infants and young children., Method: Pregnant women registered in Putuo District, Changzheng Town Community Health Service Center Child Health Clinic within the period from January to December, 2008 were enrolled into this survey, a questionnaire survey. The infants were followed up from birth to 2 years of age. The mother and child survey was conducted for 746 pairs, and 684 pairs had complete data. SAS V9.1 statistical software was used for data processing and statistical analysis., Result: The survey showed that prevalence of eczema, allergic rash, and wheezing was 27.9%, 18.9%, and 3.9%, respectively. Multivariate logistic regression analysis showed that allergy in either parent and addition of foods other than milk in infants before 4 months of age were risk factors for eczema; allergy in either parent was also risk factors for allergic rash. Exclusive breastfeeding from birth to 6 months of age was a protective factor for wheezing in infants. Other factors such as parental history of asthma, vitamin supplements to the mothers during pregnancy, mothers' special diet habits, calcium level of infants, etc. had no significant correlation with allergic disorders in infants., Conclusion: The risk factors for allergic disorders in infants included allergy in either parent and dietary factors of the infants themselves (prematurely adding other foods). Breastfeeding (for 0 - 6 months of age) was a protective factor for infants' wheezing.

Objectives: We examined the association between household solid fuel exposure and lung function in a densely populated district in urban Shanghai, China., Methods: Spirometry was performed in 12 506 subjects, aged 18 and over, residing in the Putuo District in Shanghai, China, in a cross-sectional survey. Exposure to solid fuel use at home was assessed by an administered questionnaire, estimating duration and total amount of solid fuel use at home during the lifetime., Results: After adjusting for confounders, the subjects with exposure to household solid fuel had a 1.3% (95% CI 0.57 to 2.02) decrease in forced expiratory volume in 1 s (FEV(1)) percent predicted and 3.5% (95% CI 2.74 to 4.18) decrease in forced vital capacity (FVC) percent predicted, respectively. Trends towards decreased pulmonary function measures were seen for longer duration and greater amount of household fuel use at home, in the highest compared with lowest tertile (p values for trend <0.001). We observed decrease in FEV(1) and FVC percent predicted across increase in tertile of body mass index in association with in-home solid fuel exposure., Conclusions: This study suggests that in-home solid fuel exposure is associated with reduced lung function in an urban population.

Background: Although recent research evidence suggests an association between household air pollution from solid fuel use, such as coal or biomass, and cardiovascular events such as hypertension, little epidemiologic data are available concerning such exposure effects on cardiovascular endpoints other than hypertension. We explored the association between in-home solid fuel use and self-reported diagnoses of cardiovascular endpoints, such as hypertension, coronary heart disease (CHD), stroke, and diabetes., Methods: We analyzed 14,068 Chinese adults, aged 18 years and older. Odds ratios (OR) and the corresponding 95% confidence intervals (CI) were estimated using logistic regression models for the risk of each outcome after adjusting for potential confounders., Results: The use of solid fuel in home was significantly associated with an increased risk for hypertension (OR 1.70, 95% CI 1.40 to 2.07), CHD (OR 2.58, 95% CI 1.53 to 4.32), and diabetes (OR 2.48, 95% CI 1.59 to 3.86), after adjusting for potential confounders. Compared with individuals in the lowest tertile of the duration of solid fuel exposure, those in the highest tertile of the duration of solid fuel exposure had an increased odds of hypertension (OR 1.73, 95% CI 1.45 to 2.06), stroke (OR 1.87, 95% CI 1.03 to 3.38), and diabetes (OR 3.18, 95% CI 2.11 to 4.78)., Conclusions: Our data suggest that in-home solid fuel exposure maybe associated with increased risk for hypertension, CHD, stroke, and diabetes in the Chinese adult population. Further large-scale longitudinal studies are warranted to confirm these findings.

The hepatitis C virus (HCV) non-structural (NS) 5A protein plays an essential role in the replication of the viral RNA by the membrane-associated replication complex (RC). Recently, a putative NS5A inhibitor, BMS-790052, exhibited the highest potency of any known anti-HCV compound in inhibiting HCV replication in vitro and showed a promising clinical effect in HCV-infected patients. The precise mechanism of action for this new class of potential anti-HCV therapeutics, however, is still unclear. In order to gain further insight into its mode of action, we sought to test the hypothesis that the antiviral effect of BMS-790052 might be mediated by interfering with the functional assembly of the HCV RC. We observed that BMS-790052 indeed altered the subcellular localization and biochemical fractionation of NS5A. Taken together, our data suggest that NS5A inhibitors such as BMS-790052 can suppress viral genome replication by altering the proper localization of NS5A into functional RCs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Long-term occupational exposure to cotton dust that contains endotoxin is associated with chronic respiratory symptoms and excessive decline in forced expiratory volume in 1 sec (FEV1), but the mechanisms of endotoxin-related chronic airflow obstruction remain unclear., Objective: In the current study, we examined temporal aspects of the exposure-response relationship between airborne endotoxin exposure, longitudinal change in FEV1, and respiratory symptoms in a cohort of Chinese cotton textile workers., Methods: This prospective cohort study followed 447 cotton textile workers from 1981 to 2006. at approximately 5-year intervals. We used a generalized estimating equations approach to model FEV1 level and respiratory symptoms as a function of past exposure (cumulative exposure up to the start of the most recent 5-year survey interval) and cumulative exposure (within the most recent interval) to endotoxins, after adjusting for other covariates. Models were stratified by active versus retired work status and by years employed before the baseline survey (< 5 and > or = 5 years)., Results and Conclusions: Past exposure to endotoxin was associated with reduced FEV1 level among retired cotton workers. Among all cotton workers, past exposure was more strongly associated with reduced FEV1 for those hired < 5 years before baseline than for those who were hired > or = 5 years after baseline. Recent endotoxin exposure was significantly associated with byssinosis, chronic bronchitis, and chronic cough.

Further investigation of the recently reported piperidine-4-yl-aminopyrimidine class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) has been carried out. Thus, preparation of a series of N-phenyl piperidine analogs resulted in the identification of 3-carboxamides as a particularly active series. Analogs such as 28 and 40 are very potent versus wild-type HIV-1 and a broad range of NNRTI-resistant mutant viruses. Synthesis, structure-activity relationship (SAR), clearance data, and crystallographic evidence for the binding motif are discussed., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B., (Copyright 2010 Elsevier Ltd. All rights reserved.)

An analysis of the binding motifs of known HIV-1 non-nucleoside reverse transcriptase inhibitors has led to discovery of novel piperidine-linked aminopyrimidine derivatives with broad activity against wild-type as well as drug-resistant mutant viruses. Notably, the series retains potency against the K103N/Y181C and Y188L mutants, among others. Thus, the N-benzyl compound 5k has a particularly attractive profile. Synthesis and SAR are presented and discussed, as well as crystal structures relating to the binding motifs., (2010 Elsevier Ltd. All rights reserved.)

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

The binding affinity of four palm and thumb site representative non-nucleoside inhibitors (NNIs) of HCV polymerase NS5B to wild-type and resistant NS5B polymerase proteins was determined, and the influence of RNA binding on NNI binding affinity was investigated. NNIs with high binding affinity potently inhibited HCV RNA polymerase activity and replicon replication. Among the compounds tested, HCV-796 showed slow binding kinetics to NS5B. The binding affinity of HCV-796 to NS5B increased 27-fold over a 3-h incubation period with an equilibrium Kd of 71 +/- 2 nm. Slow binding kinetics of HCV-796 was driven by slow dissociation from NS5B with a k(off) of 4.9 +/- 0.5 x 10(-4) s(-1). NS5B bound a long, 378-nucleotide HCV RNA oligonucleotide with high affinity (Kd = 6.9 +/- 0.3 nm), whereas the binding affinity was significantly lower for a short, 21-nucleotide RNA (Kd = 155.1 +/- 16.2 nm). The formation of the NS5B-HCV RNA complex did not affect the slow binding kinetics profile and only slightly reduced NS5B binding affinity of HCV-796. The magnitude of reduction of NNI binding affinity for the NS5B proteins with various resistance mutations in the palm and thumb binding sites correlated well with resistance -fold shifts in NS5B polymerase activity and replicon assays. Co-crystal structures of NS5B-Con1 and NS5B-BK with HCV-796 revealed a deep hydrophobic binding pocket at the palm region of NS5B. HCV-796 interaction with the induced binding pocket on NS5B is consistent with slow binding kinetics and loss of binding affinity with mutations at amino acid position 316.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are part of the preferred treatment regimens for individuals infected with HIV. These NNRTI-based regimens are efficacious, but the most popular NNRTIs have a low genetic barrier to resistance and have been associated with adverse events. There is therefore still a need for efficacious antiviral medicines that facilitate patient adherence and allow durable suppression of viral replication. As part of an extensive program targeted toward the discovery of NNRTIs that have favorable pharmacokinetic properties, good potency against NNRTI-resistant viruses, and a high genetic barrier to drug resistance, we focused on the optimization of a series of diaryl ether NNRTIs. In the course of this effort, we employed molecular modeling to design a new set of NNRTIs that that are active against wild-type HIV and key NNRTI-resistant mutant viruses. The structure-activity relationships observed in this series of compounds provide insight into the structural features required for NNRTIs that inhibit the replication of a wide range of mutant viruses. Selected compounds have promising pharmacokinetic profiles.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are recommended components of preferred combination antiretroviral therapies used for the treatment of HIV. These regimens are extremely effective in suppressing virus replication. Structure-based optimization of diaryl ether inhibitors led to the discovery of a new series of pyrazolo[3,4-c]pyridazine NNRTIs that bind the reverse transcriptase enzyme of human immunodeficiency virus-1 (HIV-RT) in an expanded volume relative to most other inhibitors in this class.The binding mode maintains the beta13 and beta14 strands bearing Pro236 in a position similar to that in the unliganded reverse transcriptase structure, and the distribution of interactions creates the opportunity for substantial resilience to single point mutations. Several pyrazolopyridazine NNRTIs were found to be highly effective against wild-type and NNRTI-resistant viral strains in cell culture.

Novel non-nucleoside inhibitors of HIV-RT that contain pyridazinone isosteres were prepared, and a series of triazolinones were found to be potent inhibitors of HIV replication. These compounds were active against several NNRTI-resistant virus strains. Pharmacokinetic studies indicated that inhibitor 7e has good bioavailability in rats. Several fragments of inhibitor 7c were prepared, and the binding of these compounds to HIV-RT was analyzed by surface plasmon resonance spectroscopy.

HIV reverse transcriptase (HIV-RT) contains two distinct protein domains catalyzing DNA polymerase and RNase H activities. Non-nucleoside reverse transcriptase inhibitor (NNRTI) binding to HIV-RT can affect RNase H activity. The structurally diverse NNRTIs capravirine, efavirenz, GW8248, TMC-125, and nevirapine all inhibited 5'-RNA directed HIV RNase H activity as partial inhibitors with maximal inhibition of 40-65%. Potencies of RNase H inhibition correlated with the respective potencies of DNA polymerase inhibition. Mutations in the NNRTI binding site (K103N, Y181C, Y188L, and K103N/Y181C) reduced the potency of RNase H inhibition, similar to their effects on DNA polymerase activity. The NNRTIs did not affect the activity of the isolated HIV RNase H domain. In contrast, 3'-DNA directed RNase H activity of HIV-RT was mechanistically distinct from 5'-RNA directed RNase H activity and was stimulated rather than inhibited by NNRTI binding to HIV-RT. Therefore, NNRTI binding to the polymerase domain of HIV-RT interferes with RNase H activity through a long-range effect, which is affected by the structure of the RNA:DNA hybrid substrate, but is independent of NNRTI compound structure and nucleic acid substrate sequence.

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

In order to evaluate chronic effects of long-term exposure to cotton dust on respiratory health, and the role of dust and endotoxin, longitudinal changes in lung function and respiratory symptoms were observed prospectively from 1981 to 2001 in 447 cotton textile workers, along with 472 silk textile controls. The results from five surveys conducted over the 20-yr period are reported, including standardised questionnaires, pre- and post-shift spirometric measurements, work-area inhalable dust sample collections and airborne Gram-bacterial endotoxin analysis. Cotton workers had more persistent respiratory symptoms and greater annual declines in forced expiratory volume in one second (FEV1) and forced vital capacity as compared with silk workers. After exposure cessation, in the final 5-yr period, the rate of FEV1 decline tended to slow in nonsmoking males, but not in nonsmoking females. Workers who reported byssinotic symptoms more persistently suffered greater declines in FEV1. Chronic loss in lung function was more strongly associated with exposure to endotoxin than to dust. In conclusion, the current study suggests that long-term exposure to cotton dust, in which airborne endotoxin appears to play an important role, results in substantial adverse chronic respiratory effects.

This report describes a procedure to generate enzymatically active, isolated HIV RNase H domain. In contrast to previously described preparations, the RNA cleavage activity of the untagged RNase H domain was surprisingly similar to that of the full-length HIV-RT protein. Signature cleavages at 18 and 9 nucleotides downstream of a recessed RNA 5'-end were retained with the isolated RNase H domain. Activity was strongly decreased by deletion of 3 amino acids from the C-terminus, consistent with an important structural or functional role of the C-terminal alpha-helix. A prototype N-hydroxyimide (2-hydroxy-4H-isoquinoline-1,3-dione) was found to inhibit the activity of the isolated HIV RNase H domain as well as the RNase H activity of full-length HIV reverse transcriptase. In contrast, the compound did not significantly inhibit the structurally closely related Escherichia coli RNase HI. Specific binding of N-hydroxyimide compounds to the isolated RNase H domain was observed by protein fluorescence quenching.

Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

Era is a highly conserved GTPase essential for bacterial growth. The N-terminal part of Era contains a conserved GTPase domain, whereas the C-terminal part of the protein contains an RNA- and membrane-binding domain, the KH domain. To investigate whether the binding of Era to 16S rRNA and membrane requires its GTPase activity and whether the GTPase domain is essential for these activities, the N- and C-terminal parts of the Streptococcus pneumoniae Era - Era-N (amino acids 1-185) and Era-C (amino acids 141-299), respectively - were expressed and purified. Era-C, which had completely lost GTPase activity, bound to the cytoplasmic membrane and 16S rRNA. In contrast, Era-N, which retained GTPase activity, failed to bind to RNA or membrane. These results therefore indicate that the binding of Era to RNA and membrane does not require the GTPase activity of the protein and that the RNA-binding domain is an independent, functional domain. The physiological effects of the overexpression of Era-C were assessed. The Escherichia coli cells overexpressing Era and Era-N exhibited the same growth rate as wild-type E. coli cells. In contrast, the E. coli cells overexpressing Era-C exhibited a reduced growth rate, indicating that the overexpression of Era-C inhibits cell growth. Furthermore, overexpression of era-N and era-C resulted in morphological changes. Finally, purified Era and Era-C were able to bind to poly(U) RNA, and the binding of Era to poly(U) RNA was significantly inhibited by liposome, as the amount of Era bound to the RNA decreased proportionally with the increase of liposome in the assay. Therefore, this study provides the first biochemical evidence that both binding sites are overlapping. Together, these results indicate that the RNA- and membrane-binding domain of Era is a separate, functional entity and does not require the GTPase activity or the GTPase domain of the protein for activity.

cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.

Era, an essential GTPase, plays a regulatory role in several cellular processes. The Era protein of Streptococcus pneumoniae has recently been shown to bind to 16S rRNA and the cytoplasmic membrane. However, exact locations of Era responsible for RNA- and membrane-binding were unknown. To identify the regions in Era that interact with the RNA and membrane, the C-terminal part of S. pneumoniae Era was systematically deleted while the N-terminal part, responsible for the GTPase activity of the protein, was kept intact. The resulting truncated Era proteins were purified and characterized. The C-terminal deletion of 9 or 19 amino-acid residues did not affect 16S rRNA-binding activity while further deletions of the C-terminus (29-114 amino-acid residues) abolished the activity. These results indicate that the integrity of the putative KH domain of Era, spanning the amino-acid residues between approximately 22-83 from the C-terminus, is required for 16S rRNA-binding. Furthermore, the Era proteins with a deletion up to 45 residues from the C-terminus retained membrane-binding activity, but longer deletions significantly reduced the activity. These results indicate that part of the putative KH domain is also required for membrane-binding. Thus, these results indicate for the first time that the regions critical for the membrane- and 16S rRNA-binding activities of Era overlap. The era gene with a deletion of 9 or 19 codons from its 3' terminus complemented an Escherichia coli mutant strain deficient in Era production whereas the genes with longer deletions failed to do so, thereby indicating that the KH domain is essential for Era function. Taken together, the results of this study indicate that the putative KH domain is required for 16S rRNA-binding activity and that part of the KH domain is also required for membrane-binding activity. The results also suggest that the interaction between Era and 16S rRNA is essential for bacterial growth.

Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromultimer of gpNu1 and gpA subunits. In an earlier investigation, a lethal mutation changing gpA residue 497 from lysine to aspartic acid (K497D) was found to cause a mild change in the high-affinity ATPase that resides in gpA and a severe defect in the endonuclease activity of terminase. The K497D terminase efficiently sponsored packaging of mature lambda DNA into proheads. In the present work, K497D terminase was found to have a severe defect in the cohesive end separation, or helicase, activity. Plaque-forming pseudorevertants of lambda A K497D were found to carry mutations in A that suppressed the lethality of the A K497D mutation. The two suppressor mutations identified, A E515G and A E515K, affected residue 515, which is located near the putative P-loop of gpA. A codon substitution study of codon 515 showed that hydrophobic and basic residues suppress the K497D defect, but hydrophilic and acidic residues do not. The E515G change was demonstrated to reverse the endonuclease and helicase defects caused by the K497D change. Moreover, the gpA K497D E515G enzyme was found to have kinetic constants for the high-affinity ATPase center similar to those of the wild type enzyme, and the endonuclease activity of the K497D E515G enzyme was stimulated by ATP to an extent similar to the ATP stimulation of the endonuclease activity of the wild type enzyme., (Copyright 2000 Academic Press.)

Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging., (Copyright 2000 Academic Press.)