Association of long-term indoor exposure to fine particles with pulmonary effects in Northern Taiwan.

An association between short-term indoor exposure to fine particles (PM2.5) and acute respiratory effects has been reported. It is still unclear whether long-term indoor exposure to PM2.5 is associated with pulmonary events. This study recruited 1023 healthy adult homeworkers to conduct a prospective observational study from 2010 to 2021. Four repeated home visits per year were conducted for each participant to measure 24-hour PM2.5 and peak expiratory flow rate (PEFR) and to collect blood samples for absolute eosinophil count (AEC) and carcinoembryonic antigen (CEA) analysis. Additionally, a questionnaire related to personal characteristics, health status and home characteristics was conducted for each participant. The mixed-effects models showed a significant association of PM2.5 with increased CEA and AEC and decreased % predicted PEFR. No significant association between low-level PM2.5 exposure (10-year mean level < 10 µg/m3) and adverse pulmonary effects was observed. The present study concluded that long-term indoor exposure to PM2.5 at a concentration higher than 10 µg/m3 was associated with adverse pulmonary effects among healthy adult homeworkers.

Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters.

BACKGROUND & AIMS: We aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS). METHODS: Fifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies. RESULTS: HBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome. CONCLUSIONS: Higher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome. LAY SUMMARY: HBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing.

Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated "base editors" (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.

Long-term indoor air conditioner filtration and cardiovascular health: A randomized crossover intervention study.

The association of short-term air pollution filtration with cardiovascular health has been documented. However, the effect of long-term indoor air conditioner filtration on the association between air pollution and cardiovascular health is still unclear. We recruited 200 homemakers from Taipei and randomly assigned 100 of them to air filtration or control intervention; six home visits were conducted per year from 2013 to 2014. The participants under air filtration intervention during 2013 were reassigned to control intervention in 2014. The air pollution measurements consisted of particulate matter less than or equal to 2.5µm in diameter (PM2.5) and total volatile organic compounds (VOCs); blood pressure was monitored for each participant during each visit. The following morning, blood samples were collected after air pollution monitoring. The blood samples were used to analyze biological markers, including high sensitivity-C-reactive protein (hs-CRP), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and fibrinogen. Household information, including cleaning, cooking, and air conditioning, was collected by a questionnaire. Mixed-effects models were used to investigate the associations among air pollution measurements, blood pressure and biological markers. The results showed that increased levels of PM2.5 and total VOCs were associated with increased hs-CRP, 8-OHdG and blood pressure. The health variables were higher among participants in the control intervention phase than among those in the air filtration intervention phase. We concluded that air pollution exposure was associated with systemic inflammation, oxidative stress and elevated blood pressure. The long-term filtration of air pollution with an air conditioner filter was associated with cardiovascular health of adults.

Effect of welding fume on heart rate variability among workers with respirators in a shipyard.

Welding fume exposure is associated with heart rate variability (HRV) reduction. It is still unknown whether respirator can reduce effect of welding fume on HRV among welding workers in a shipyard. We recruited 68 welding workers with respirator and 52 welding workers without respirator to measure HRV indices, including standard deviation of normal-to-normal intervals (SDNN) and square root of the mean squared differences of successive intervals (r-MSSD) by ambulatory electrocardiographic (ECG). Personal exposure to particulate matter less than or equal to 2.5 µm in diameter (PM2.5) was measured by a dust monitor. The association between 5-minute mean PM2.5 and log10-transformed HRV indices was analyzed by mixed-effects models. We found 5-minute mean PM2.5 was associated with 8.9% and 10.3% decreases in SDNN and r-MSSD. Effect of PM2.5 on HRV indices was greatest among workers without respirator {SDNN: 12.4% (95% confidence interval = -18.8--6.9); r-MSSD: 14.7% (95% confidence interval = -20.8--8.6)}. Workers with respirator showed slight decreases in HRV indices {SDNN: 2.2% (95% confidence interval = -6.3--1.9); r-MSSD: 4.0% (95% confidence interval = -6.4--1.6)}. We conclude that respirator use reduces the effect of PM2.5 exposure on HRV among workers performing welding in a shipyard.

Effects of commuting mode on air pollution exposure and cardiovascular health among young adults in Taipei, Taiwan.

The association between traffic-related air pollution and adverse cardiovascular effects has been well documented; however, little is known about whether different commuting modes can modify the effects of air pollution on the cardiovascular system in human subjects in urban areas with heavy traffic. We recruited 120 young, healthy subjects in Taipei, Taiwan. Each participant was classified with different commuting modes according to his/her own commuting style. Three repeated measurements of heart rate variability (HRV) indices {standard deviation of NN intervals (SDNN) and the square root of the mean of the sum of the squares of differences between adjacent NN intervals (r-MSSD)}, particulate matter with an aerodynamic diameter ≤ 2.5 µm (PM2.5), temperature, humidity and noise level were conducted for each subject during 1-h morning commutes (0900-1000 h) in four different commuting modes, including an electrically powered subway, a gas-powered bus, a gasoline-powered car, and walking. Linear mixed-effects models were used to investigate the association of PM2.5 with HRV indices. The results showed that decreases in the HRV indices were associated with increased levels of PM2.5. The personal exposure levels to PM2.5 were the highest in the walking mode. The effects of PM2.5 on cardiovascular endpoints were the lowest in the subway mode compared to the effects in the walking mode. The participants in the car and bus modes had reduced effects on their cardiovascular endpoints compared to the participants in the walking mode. We concluded that traffic-related PM2.5 is associated with autonomic alteration. Commuting modes can modify the effects of PM2.5 on HRV indices among young, healthy subjects.

The effect of essential oil on heart rate and blood pressure among solus por aqua workers.

BACKGROUND: Aromatherapy is widely used around the world for stress relief. Whether exposure to essential oil increases the risk of cardiovascular events is still unknown. The purpose of this study was to investigate the effect of essential oil on heart rate and blood pressure among solus por aqua (spa) workers. METHODS: We recruited 100 healthy workers from various spa centres in Taipei, Taiwan. Between July and August of 2010, three repeated measurements - resting heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) - were taken of each spa worker in our study room. Participants were exposed to essential oil vapour generated from an ultrasonic atomizer in the study room for two consecutive hours. The total volatile organic compound (VOC) level in the study room was measured during the study period. We used a linear mixed-effect model to determine the association between the total VOC level and the participants' HR, SBP, and DBP. RESULTS: For the times from 15 to 60 min after start of exposure, we found that the VOC level was significantly associated with reduced 15-min mean BP and HR. After exposure for more than 1 hour, from 75 to 120 min after start of exposure, we found that the VOC levels were associated with increased 15-min mean BP and HR. CONCLUSIONS: Exposure to essential oil for 1 hour was found to be an effective method of relaxation, as indicated by decreases in the HR and BP. Prolonged exposure for longer than 1 hour to essential oils may be harmful to cardiovascular health among spa workers.

Peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection.

BACKGROUND: Feline infectious peritonitis (FIP) is a lethal immune-mediated disease caused by feline coronavirus (FCoV). Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2) sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated. METHODS: Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection. RESULTS: The results demonstrated that peptide (FP5) at concentrations below 20 µM inhibited viral replication by up to 97%. The peptide (FP5) exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α), and a significant synergistic antiviral effect was observed. CONCLUSION: Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.

Reducing indoor air pollution by air conditioning is associated with improvements in cardiovascular health among the general population.

Indoor air pollution is associated with cardiovascular effects, however, little is known about the effects of improving indoor air quality on cardiovascular health. The aim of this study was to explore whether improving indoor air quality through air conditioning can improve cardiovascular health in human subjects. We recruited a panel of 300 healthy subjects from Taipei, aged 20 and over, to participate in six home visits each, to measure a variety of cardiovascular endpoints, including high sensitivity-C-reactive protein (hs-CRP), 8-hydroxy-2'-deoxyguanosine (8-OHdG), fibrinogen in plasma and heart rate variability (HRV). Indoor particles and total volatile organic compounds (VOCs) were measured simultaneously at the participant's home during each visit. Three exposure conditions were investigated in this study: participants were requested to keep their windows open during the first two visits, close their windows during the next two visits, and close the windows and turn on their air conditioners during the last two visits. We used linear mixed-effects models to associate the cardiovascular endpoints with individual indoor air pollutants. The results showed that increases in hs-CRP, 8-OHdG and fibrinogen, and decreases in HRV indices were associated with increased levels of indoor particles and total VOCs in single-pollutant and two-pollutant models. The effects of indoor particles and total VOCs on cardiovascular endpoints were greatest during visits with the windows open. During visits with the air conditioners turned on, no significant changes in cardiovascular endpoints were observed. In conclusion, indoor air pollution is associated with inflammation, oxidative stress, blood coagulation and autonomic dysfunction. Reductions in indoor air pollution and subsequent improvements in cardiovascular health can be achieved by closing windows and turning on air conditioners at home.

Identification of a minimal peptide derived from heptad repeat (HR) 2 of spike protein of SARS-CoV and combination of HR1-derived peptides as fusion inhibitors.

The heptad repeats (HR1 and HR2) of the spike protein of SARS-CoV are highly conserved regions forming a critical 6-helix bundle during the fusion step of virus entry and are attractive targets of entry inhibitors. In this study, we report that a minimal HR2 peptide, P6 of 23-mer, can block the fusion of SARS-CoV with an IC(50) of 1.04+/-0.22 microM. This finding supports the structural prediction of the deep groove of HR1 trimer as a target for fusion inhibitors, and suggests P6 as a potential lead peptide for future drug development. Moreover, combination of an HR-1 peptide, N46, and its mutated version, N46eg, shows synergistic inhibition with an IC(50) of 1.39+/-0.05 microM and combination index of 0.75+/-0.15, suggesting a common strategy to achieve promising inhibition by HR1 peptide for other class I envelope viruses.

A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles.

Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.

Immunofluorescence assay for detection of the nucleocapsid antigen of the severe acute respiratory syndrome (SARS)-associated coronavirus in cells derived from throat wash samples of patients with SARS.

An antigen detection assay for severe acute respiratory syndrome (SARS) coronavirus was established in this study by an indirect immunofluorescence test, which utilized cells derived from throat wash samples of patients with SARS and a rabbit serum that recognized the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) but not that of other human coronavirus tested. It detected SARS-CoV in 11 of 17 (65%) samples from SARS patients as early as day 2 of illness but in none of the 10 samples from healthy controls. Compared with other diagnostic modalities for detecting SARS-CoV, this assay is simpler, more convenient, and economical. It could be an alternative for early and rapid diagnosis, should SARS return in the future.

Temporal relationship of viral load, ribavirin, interleukin (IL)-6, IL-8, and clinical progression in patients with severe acute respiratory syndrome.

Although viral replication and overwhelming immune responses are believed to contribute to the progression of severe acute respiratory syndrome (SARS), little is known about the temporal relationship between viral load, ribavirin, proinflammatory cytokines, and clinical progression. We report that ribavirin was not effective in reducing the SARS coronavirus load in 3 of 8 probable cases studied and that elevated levels of interleukin (IL)-6 and IL-8 subsequent to the peak viral load were found in 8 and 6 cases, respectively. The nadir lymphocyte count during lymphopenia, the peak level of lactate dehydrogenase, and the peak density of pulmonary infiltrates lag further behind the peak viral load by a median of 4, 5, and 3.5 days, respectively. These findings provide important information for therapeutic strategies to treat SARS.

Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to be transmitted primarily through dispersal of droplets, but little is known about the load of SARS-CoV in oral droplets. We examined oral specimens, including throat wash and saliva, and found large amounts of SARS-CoV RNA in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/mL) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/mL) from all specimens of 17 consecutive probable SARS case-patients, supporting the possibility of transmission through oral droplets. Immunofluorescence study showed replication of SARS-CoV in the cells derived from throat wash, demonstrating the possibility of developing a convenient antigen detection assay. This finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for SARS diagnosis.