Overexpression of USP8 inhibits inflammation and ferroptosis in chronic obstructive pulmonary disease by regulating the OTUB1/SLC7A11 signaling pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a familiar disease, and owns high morbidity and mortality, which critically damages the health of patients. Ubiquitin-specific peptidase 8 (USP8) is a pivotal protein to join in the regulation of some diseases. In a previous report, it was determined that USP8 expression is down-regulated in LPS-treated BEAS-2B cells, and USP8 restrains inflammatory response and accelerates cell viability. However, the regulatory roles of USP8 on ferroptosis in COPD are rarely reported, and the associated molecular mechanisms keep vague. OBJECTIVE: To investigate the regulatory functions of USP8 in COPD progression. MATERIAL AND METHODS: The lung functions were measured through the Buxco Fine Pointe Series Whole Body Plethysmography (WBP). The Fe level was tested through the Fe assay kit. The protein expressions were assessed through western blot. The levels of tumor necrosis -factor-α, interleukin 6, and interleukin 8 were evaluated through enzyme-linked immunosorbent serologic assay. Cell viability was tested through CCK-8 assay. RESULTS: In this work, it was discovered that overexpression of USP8 improved lung function in COPD mice. In addition, overexpression of USP8 repressed ferroptosis by regulating glutathione peroxidase 4 and acyl-CoA synthetase long-chain family 4 expressions in COPD mice. Overexpression of USP8 suppressed inflammation in COPD mice. Furthermore, overexpression of USP8 suppressed ferroptosis in COPD cell model. At last, it was verified that overexpression of USP8 accelerated ubiquitin aldehyde-binding protein 1 (OTUB1)/solute carrier family 7 member 11 (SLC7A11) pathway. CONCLUSION: This study manifested that overexpression of USP8 restrained inflammation and ferroptosis in COPD by regulating the OTUB1/SLC7A11 signaling pathway. This discovery hinted that USP8 could be a potential target for COPD treatment.

Epidemiological and clinical characteristics of vaccinated COVID-19 patients: A meta-analysis and systematic review.

OBJECTIVE: With the global epidemic of coronavirus disease 2019 (COVID-19), vaccination rates are increasing globally. This study evaluated the relevant clinical manifestations of vaccinated COVID-19 patients. METHODS: We searched carefully in 11 databases such as PubMed, Embase, Scopus, Cochrane Library, Web of Science, Ovid, China National Knowledge Infrastructure Database, Wan Fang Data, Sinomed, VIP Database, and Reading Showing Database up to 26 March 2022. To search for articles that have described the characteristics of vaccinated patients including epidemiological and clinical symptoms. Statistical analysis of the extracted data using STATA 14.0. RESULTS: A total of 58 articles and 263,708 laboratory-confirmed COVID-19 patients were included. Most of the patients in the vaccinated group had more asymptomatic infection and fewer severe illnesses. There were significant differences in ethnicity, and strain infected with COVID-19, and comorbidities (hyperlipidemia, diabetes, obesity, kidney disease, immunocompromised, cardiovascular disease, and tumor) and symptoms (fever, cough, gastrointestinal symptoms, neurological symptoms, and dysgeusia/anosmia) between vaccinated group and unvaccinated group. Oxygen support, use of steroid, days in hospital, hospital treatment, ICU treatment, death, and poor prognosis were also significantly different. CONCLUSION: Compared with the vaccinated group, patients in the unvaccinated group had a more severe clinical manifestations. Vaccines are also protective for infected people.

Ubiquitin-specific protease 8 inhibits lipopolysaccharide-triggered pyroptosis of human bronchial epithelial cells by regulating PI3K/AKT and NF-κB pathways.

Asthma, characterized by dysfunction of airway epithelial cells, is regarded as a chronic inflammatory disorder in the airway. Ubiquitin-specific protease 8 (USP8) belongs to ubiquitin proteasome system and mediates the stability of E3 ligases. The anti-inflammatory effect of USP8 has been widely investigated in distinct diseases, while the role of USP8 in asthma remains elusive. Firstly, human bronchial epithelial cells (BEAS-2B) were treated with lipopolysaccharide, which reduced the cell viability of BEAS-2B and induced the secretion of lactate dehydrogenase (LDH). Moreover, the expression of USP8 was downregulated in BEAS-2B post lipopolysaccharide treatment. Secondly, overexpression of USP8 enhanced cell viability of lipopolysaccharide-treated BEAS-2B, and reduced the LDH secretion. USP8 overexpression also attenuated lipopolysaccharide-induced upregulation of TNF-α, IL-6, and IL-1ß in BEAS-2B. Thirdly, lipopolysaccharide treatment promoted the expression of NLRP3 (NLR Family Pyrin Domain Containing 3), N-terminal domain of gasdermin D (GSDMD-N), caspase-1, IL-1ß, and IL-18 in BEAS-2B, which was inhibited by USP8 overexpression. Lastly, USP8 overexpression decreased the phosphorylation of NF-κB, while it increased the phosphorylation of PI3K and AKT in lipopolysaccharide-treated BEAS-2B. In conclusion, USP8 inhibited lipopolysaccharide-triggered inflammation and pyroptosis in human bronchial epithelial cells by activating PI3K/AKT signaling and inhibiting NF-κB signaling pathway.

Hydroa Vacciniforme-like Lymphoproliferative disorder in an adult invades the liver and bone marrow with clear pathological evidence: a case report and literature review.

BACKGROUND: Hydroa Vacciniforme-like Lymphoproliferative Disorder (HV-LPD) is the name given to a group of Epstein-Barr virus (EBV)-associated diseases. It resembles hydroa vacciniforme (HV), the rarest form of photosensitivity, and is a T-cell disorder associated with an Epstein-Barr virus infection. The majority of diagnosed cases occur in East Asia and South America. It is rare in the United States and Europe. Multiple studies have revealed the clinical manifestation of an enlarged liver, but no gold standard such as pathology has yet supported this as a clinical sign of HV-LPD. CASE PRESENTATION: Here, we report a case of a 34-year-old Asian female with definite liver invasion. The patient had complained of a recurring facial rash for many years. The patient was admitted to the hospital because of an enlarged liver. After hospitalization, she was given an EB virus nucleic acid test. The EB virus nucleic acid test was positive, and pathological examination suggested that HV-LPD had invaded the skin, bone marrow, and liver. After being given antiviral treatment, the patient's symptoms were mitigated. CONCLUSIONS: Our case confirms the liver damage was caused by HV-LPD and the effectiveness of antiviral treatment.

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

Development and Evaluation of a Universal and Supersensitive NS1-Based Luciferase Immunosorbent Assay to Detect Zika Virus-Specific IgG.

Zika virus (ZIKV) causes rash, moderate fever, conjunctivitis, and arthralgia, and has serious connection with neurological complications; therefore, it is a major threat to public health. A rapid and supersensitive method for detecting anti-ZIKV antibodies in humans and animals is thus urgently required. Here, we report an NS1-based luciferase immunosorbent assay (LISA), developed to detect ZIKV-specific IgG. Fusion proteins including a reporter Nano-luciferase (NLuc) and various fragments of ZIKV NS1 protein were expressed in 293 T cells. LISA was performed using the above cell lysates containing the expressed fusion proteins. Sample panels of humans and animals infected with ZIKV were examined for sensitivity of LISA, relative to those of ZIKV RT-PCR, commercial NS1-based ELISA, and micro-neutralization (MN) assays. Specificity and potential cross-reactivity were also evaluated using various convalescent serum samples derived from patients infected with dengue virus (DENV), Japanese encephalitis virus (JEV), and hepatitis C virus (HCV). Results indicated the optimal antigenic domain for anti-ZIKV IgG detection was located within 172-352 amino acids (aa) of ZIKV NS1 protein. NS1-based LISA performs better than commercial ELISA in anti-ZIKV IgG detection. LISA was shown to be at least fourfold more sensitive than commercial ELISA, and could detect anti-ZIKV IgG in various animal hosts without the need of species-specific labeled antibody. This novel assay is potentially useful for the rapid and sensitive detection of anti-ZIKV IgG in human and animal samples.

Expression profiles of long non-coding RNA in mouse lung tissue exposed to radon.

Long non-coding RNAs (lncRNA) exert biological functions by interacting with RNAs, proteins, and DNA. Although lung damage associated with radon exposure was attributed to disturbances in microRNA and protein expression, the influence of radon on lncRNA is at present not known. The aim of this study was to (1) examine the effect of radon on lncRNA-mediated expression of transcription factors in mRNA in mouse lung tissue and (2) determine potential function and targets. Female BALB/c mice were divided into two groups: control and radon exposure to approximately 100,000 Bq/m3 (equivalent up to 60 working level month, WLM).RNA was extracted from lung tissue and used for high through-put lncRNA microarray analysis. A total of 1256 lncRNA transcripts were differentially expressed between the two groups of mice. Among these, the top 200 lncRNA-mRNA sets, with fold change of >2 and p-value <.05, were selected for KEGG analysis. Functional analysis via bioinformatics prediction in this study also suggested involvement of ErbB and Notch pathways in radon-induced mouse pulmonary injury. The results from immunohistochemical and Western blot analysis indicated that EbB2 and k-Ras protein expressions were significantly increased. In conclusion, approximately 1,000 dysregulated lncRNA transcripts were found in radon-exposed mice and these lncRNA may play an important role in lung damage following radon exposure. The observations in this study also suggested that ErbB2 and Notch pathways are activated and may be involved in radon-induced pulmonary toxicity.

Neutralization mechanism of human monoclonal antibodies against Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV.

Source contributions to total concentrations and carcinogenic potencies of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in ambient air: a case study in Suzhou City, China.

The potential source categories and source contributions of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in ambient air from Suzhou City, China, were performed by principal component analysis-multiple linear regression (PCA-MLR) and positive matrix factorization (PMF). The carcinogenic potencies of PCDD/Fs were quantitatively apportioned based on the positive matrix factorization-toxic equivalent concentration (PMF-TEQ) method. The results of the present study were summarized as follows. (1) The total concentrations and toxic equivalent concentrations of PCDD/Fs (∑PCDD/Fs and TEQ) in ambient air from Suzhou City were 1.34-42.80 pg N m-3 and 0.081-1.22 pg I-TEQ N m-3, respectively. (2) PCA-MLR suggested that industrial combustion (IC), electric arc furnaces (EAFs) and secondary aluminum smelters (ALSs), unleaded gas-fueled vehicle sources (UGFVs), ALSs, and hazardous solid waste incinerators (HSWIs) could be the primary PCDD/F contributors, accounting for 13.2, 16.7, 35.5, 19.4, and 15.2% of ∑PCDD/Fs, respectively. (3) PMF and PMF-TEQ indicated that EAFs (carbon steel), UGFVs, IC, ALSs, municipal solid waste incinerators (MSWIs) and hospital waste incinerators (HWIs), and HSWIs contributed 10.9, 10.9, 42.8, 11.3, 10.7, and 13.4% to ∑PCDD/Fs, but contributed 8.3, 12.3, 50.3, 12.7, 6.0, and 10.4% to carcinogenic potencies of PCDD/Fs. This study was the first attempt to quantitatively apportion the source-specific carcinogenic potencies of PCDD/Fs in ambient air.

PCDD/Fs distribution characteristics and health risk assessment in fly ash discharged from MSWIs in China.

This study provided distribution and health risk information of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in fly ash from 4 municipal solid waste incinerators (MSWIs) in four seasons from four sites, including Zhengzhou City in Henan Province, Chuzhou City in Anhui Province, Jilin City in Jilin Province and Zibo City in Shandong Province. The toxic equivalent concentration (I-TEQ) values of PCDD/Fs ranged from 0.0707 to 0.7742ng I-TEQ/g, and no identical sequence occurred during four seasons in different sampling sites. The stabilization process might efficiently reduce the content and toxicology of PCDD/Fs in fly ash. The value of PCDD/PCDF in fly ash ranged from 0.145 to 0.787 after solidification. The characteristic index (DCI) of 2,3,4,7,8-P5CDF was 0.803 with 6.6% under 95% probability for fly ash samples discharged from MSWIs. The 95th percentile carcinogenic risks (CRs) for onsite workers were lower than the threshold value (10-5), suggesting that the cancer risk levels of PCDD/Fs in fly ash for onsite workers were acceptable. The 95th percentile non-carcinogenic risks (non-CRs) for onsite workers were lower than 1, suggesting no obvious non-carcinogenic effect was developed for onsite workers. This paper provide an overview information on the distribution of PCDD/Fs in fly ash during four seasons, and it could be used as an important fingerprint to distinguish the fly ash sources. Thus, the research could provide basic information for fly ash management in environment.

A fatal yellow fever virus infection in China: description and lessons.

Yellow fever (YF) is a viral disease endemic to the tropical regions of Africa and South America. An outbreak of YF has been occurring in Angola, since the beginning of 2016. In March 2016, a 32-year-old Chinese man who returned from Angola was hospitalized and diagnosed with the first case of imported YF in China. Clinical observations, blood viral RNA detection, serological testing and treatments for the patient were performed daily. The virus was isolated in Vero cells, and the complete viral genome was sequenced and analyzed using the next-generation genomic sequencing platform. The patient presented with hemorrhagic fever, jaundice and oliguria at day 3 after onset, which rapidly progressed to multisystem organ failure with extremely elevated liver, pancreatic and myocardial enzymes. The patient died despite the intensive supportive treatments that were performed. A liver biopsy showed severe and multilobular necrosis. Viral RNA was detectable throughout the clinical course of the disease. Whole-genomic sequence analysis revealed that the virus belongs to the Angola71 genotype. Although the virus has been circulating in Angola for 45 years, only 14 amino-acid substitutions and no amino-acid changes were observed in the membrane and envelope proteins compared with the virus collected in 1971. The presence of this imported YF case in China indicated that with the increase in business travel among countries, YF outbreaks in Africa can lead to the international spread of the disease. The production and use of YF vaccines is, therefore, an urgent issue.

Composition profiles and health risk of PCDD/F in outdoor air and fly ash from municipal solid waste incineration and adjacent villages in East China.

In present study, composition profiles and health risk of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in outdoor air and fly ash from domestic waste treatment center (DWTC) were studied. In addition, the composition profiles and health risk of PCDD/F in outdoor air from adjacent villages were researched and used to quantitatively analyze the difference between onsite workers and adjacent villagers. Moreover, the difference between old intake method and new inhalation dosimetry method in the process of assessing the health risk of PCDD/Fs in outdoor air was quantitatively compared and analyzed. The results of this study were summarized as follows. (1) The 95th percentile carcinogenic risk (CR) and non-carcinogenic risk (non-CR) for onsite workers and adjacent villagers were much lower than the threshold values of 10(-6) and 1.0, respectively, suggesting no potential health risk. (2) The 95th percentile CR for onsite workers was 1.27×10(-8) and was 64.8 times higher than that of adjacent villagers (1.99×10(-10)). (3) The 95th percentile non-CR for onsite workers and adjacent villagers were 1.37×10(-4) and 1.31×10(-7), respectively. (3) Accidental ingestion of fly ash was the largest contributor to CR and non-CR for onsite workers, contributing 62.98% and 64.04% to CR and non-CR, respectively. (4) The CR and non-CR of PCDD/Fs in outdoor air for onsite workers and adjacent villagers which calculated by old intake method was much higher than the results from new inhalation dosimetry method. The results quantitatively showed the levels and potential risks of PCDD/Fs posed by a DWTC site, which can be helpful to predict the influence from DWTC sites and promote the management of DWTC in China.

Analysis of the miRNA-mRNA networks in malignant transformation BEAS-2B cells induced by alpha-particles.

The aim of this study was to determine the toxicity induced by irradiation with alpha-particles on malignant transformation of immortalized human bronchial epithelial cells (BEAS-2B) using miRNA-mRNA networks. The expression of BEAS-2B cells was determined by measuring colony formation, mtDNA, mitochondrial membrane potential (MMP), and ROS levels. Changes in BEAS-2B cell gene expression were observed and quantified using microarrays that included an increase in 157 mRNA and 20 miRNA expression and a decrease in 77 mRNA and 48 miRNA. Bioinformatic software was used to analyze these different mRNA and miRNA, which indicated that miR-107 and miR-494 play an important role in alpha-particles-mediated cellular malignant transformation processes. The pathways related to systemic lupus erythematosus, cytokine-cytokine receptor interaction, MAPK signaling pathway, regulation of actin cytoskeleton, and cell adhesion molecules (CAMs) were stimulated, while those of ribosome, transforming growth factor (TGF)-beta signaling pathway, and metabolic pathways were inhibited. Data suggest that miRNA and mRNA play a crucial role in alpha-particles-mediated malignant transformation processes. It is worth noting that three target genes associated with lung cancer were identified and upregulated PEG 10 (paternally expressed gene 10), ARHGAP26, and IRS1.

Synergistic effect of radon and sodium arsenite on DNA damage in HBE cells.

Human epidemiological studies showed that radon and arsenic exposures are major risk factors for lung cancer in Yunnan tin miners. However, biological evidence for this phenomenon is absent. In this study, HBE cells were exposed to different concentrations of sodium arsenite, different radon exposure times, or a combination of these two factors. The results showed a synergistic effect of radon and sodium arsenite in cell cytotoxicity as determined by cell viability. Elevated intracellular ROS levels and increased DNA damage indexed by comet assay and γ-H2AX were detected. Moreover, DNA HR repair in terms of Rad51 declined when the cells were exposed to both radon and sodium arsenite. The synergistic effect of radon and sodium arsenite in HBE cells may be attributed to the enhanced DSBs and inhibited HR pathway upon co-exposure.

Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon.

Radon has long been recognized as a human carcinogen leading to lung cancer, but the underlying mechanisms remain obscure. Recent studies have shown that the let-7 microRNA and K-ras play an important role in the development of various cancers. However, the exact role between let-7 and K-ras in radon induced lung damage has not been explored so far. In the present study, wistar rats and human bronchial epithelial (HBE) cells were long-term exposed to radon, and then alterations in histological pathology of rat lung tissue, ROS, antioxidant enzymes activities and clonogenic formation in HBE cells, as well as changes in let-7 and K-ras expression were determined to observe the adverse effects induced by radon. The results showed that long-term exposure to radon produced severe lung damage in rats, significantly increased ROS production and clonogenic formation ratios and decreased SOD activities in HBE cells. In addition, an obvious down-regulation of let-7 and up-regulation of K-ras were also revealed both in mRNA and in protein level in lung tissue of rats and HBE cells exposed to radon. Furthermore, a significant down-regulation of K-ras was then confirmed in both let-7b-3p and let-7a-2-3p transfected HBE cells. Taken together, the present results propose an involvement of let-7 microRNA and K-ras in radon induced lung damage both in vivo and in vitro, which may thus be of potential value in early diagnosis and therapy of radon-induced lung tumorgenesis.

Oxidative DNA damage is involved in cigarette smoke-induced lung injury in rats.

OBJECTIVE: Reactive oxygen species (ROS) induced by exogenous toxicants are suggested to be involved in carcinogenesis by oxidative modification of DNA. 8-Hydroxyl-2-deoxyguanosine (8-OHdG) has been considered as a reliable biomarker for oxidative DNA damage both in vivo and in vitro studies. But the effect of smoking on oxidative damage has not yet been fully elucidated. METHODS: Wistar rats were exposed to cigarette smoke at concentrations of 20 and 60 % for 30 min, twice/day for 45 weeks. Then the histopathology of lung tissues, levels of ROS, 8-OHdG, and total antioxidant (T-AOC), expression of DNA repair enzymes, e.g. 8-oxyguaine DNA glycosylase (OGG1), and MutThomolog 1 (Oxidized Purine Nucleoside Triphosphatase, MTH1) were determined in urine, peripheral blood lymphocytes, and lung tissue. RESULTS: The results showed that long-term cigarette smoke exposure can cause obvious damages of lung tissue in rats. In addition, a significant and cigarette smoke concentration-dependent increase in ROS and 8-OHdG were observed compared with the non-exposed control rats. In contrast, the expression of OGG1 and MTH1, and T-AOC levels were obviously decreased after long-term exposure to cigarette smoke. CONCLUSION: These findings indicate that long-term exposure to cigarette smoker increases ROS levels, decreases total antioxidant capacity, and interferes DNA repair capacity that eventually induces oxidative DNA damage, which appears to play an important role in cigarette smoke-induced lung injury in rats, and determination of 8-OHdG levels might be a useful method for monitoring oxidative damage in cigarette smokers.

Oxidative damage in various tissues of rats exposed to radon.

Oxidative damage can be induced by many environmental stressors. 8-Hydroxydeoxyguanosine (8-OHdG) has been used as a biomarker of oxidative DNA damage in both in vitro and in vivo studies. In the present study, Wistar rats were exposed to radon gas at a concentration of 100,000Bq/m(3) for 12 h/d for 30, 60, and 120 d, equivalent to cumulative doses of 60, 120, and 240 working level months (WLM), respectively. Changes in levels of 8-OHdG, reactive oxygen species (ROS), and total antioxidant (T-AOC), as well as expressions of some DNA repair enzymes such as 8-oxoguanine DNA glycosylase (OGG1) and MutT homolog 1 (oxidized purine nucleoside triphosphatase, MTH1), were determined in rat urine, peripheral blood lymphocytes, and lung after exposure to radon. The results revealed an increase in 8-OHdG and ROS levels, a decrease in T-AOC levels, and reduced OGG1 and MTH1 expression levels. The elevated amount of 8-OHdG in urine or lymphocytes was positively correlated with the cumulative exposure dose, whereas OGG1 and MHT1 expression levels in lung were inversely correlated with cumulative exposure dose. These findings indicate that oxidative damage induced by radon may be involved in radon-induced carcinogenesis.

Cigarette smoke-induced failure of apoptosis resulting in enhanced neoplastic transformation in human bronchial epithelial cells.

The lack of apoptotic pathways may lead to undesirable cell survival and proliferation, which are recognized hallmarks of cancer. It is well known that exposure to cigarette smoke induces DNA lesions in pulmonary cells. At present, it is not fully elucidated whether these lesions are repaired to restore normal functions or induce apoptosis. In order to examine the role of apoptosis in smoking-induced effects, immortalized human bronchial epithelial cells (BEAS-2B) were exposed to cigarette smoke and examined for parameters associated with apoptosis and neoplastic transformation. Our results indicated a significant reduction in apoptosis and enhanced neoplastic transformation and decreased mitochondrial membrane potential Δψm of mitochondria compared to control cells. Time-course experiments revealed increased aberrant methylation of CpG islands of RAS-associated domain family protein 1A (RASSF1A) and O (6)-methylguanine-DNA-methyltransferase (MGMT). The activities were downregulated and repair of DNA adducts was inhibited. Our observations suggested that although cigarette smoke-induced damage in BEAS-2B cells after chronic exposure is not necessarily lethal, as evidenced by cell viability, the protein expression levels of caspase-3 showed a decrease in the S20 passage (metaphase) but subsequently increased from S30 to S40 (anaphase). Survivin expression was significantly changed in S5 cells, and this rise was maintained until S40. Our data suggest that the potency of cigarettes as carcinogens may be due to their ability to induce aberrant gene expression and failure to trigger apoptosis leads to subsequent neoplastic transformation.

Evolution of viral RNA in a Chinese patient to interferon/ribavirin therapy for hepatitis C.

OBJECTIVE: The combination of interferon (IFN) and ribavirin (RBV) is the standard therapy for hepatitis C virus (HCV) infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet limited. This study aimed to investigate the mechanism of the poor response in a case of HCV genotype 2a infection. METHODS: We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to IFN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 (JFH1) replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient's sequence from the pre/post-treatment, and the chimeric replicons' susceptibility to IFN were evaluated by relative Gausia Luciferase activity. RESULTS: The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. CONCLUSIONS: During the course of the chronic infection, HCV population seems to be adapted to the patient's immunological system, and further to be selected by combination of IFN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region (ISDR) of NS5A during chronic infection and IFN/RBV therapy.