A Real-Time Respiration Monitoring System Using WiFi Sensing Based on the Concentric Circle Model.

This paper proposes and experimentally validates a novel concentric circle (CC) model for indoor WiFi sensing. By setting the transmitter and receiver together, the perception model becomes concentric circles with equal spacing, eliminating the blind zone and unequal radial sensitivity problems of the Fresnel zone (FZ) model. Then a human respiratory monitoring system is developed based on this model, which executes the following steps: (1) Principal component analysis (PCA) is applied to the channel state information ratio (CSIR) as a preprocessing to extract the components related to human activities. (2) Human presence and respiratory signal detection are adopted to improve monitoring accuracy. (3) The Doppler respiratory frequency is extracted to calculate the respiratory rate. Experimental results show that the CC model achieves high accuracy in velocity measurement with an error of less than 0.4 cm/s. The respiration monitoring system can accurately monitor human respiration with an error of less than 0.7 bpm within 6 m.

A Vocal Hygiene Program for Mitigating the Effects of Occupational Vocal Demand in Primary School Teachers.

PURPOSE: Teachers are occupational voice users with significant vocal demand. This study examined if a vocal hygiene program could mitigate the effects of occupational vocal demand in primary school teachers across 1 month. METHOD: Sixty female teachers participated, with 30 in an experimental group receiving vocal hygiene education plus daily home practice for 1 month and 30 in a control group with no intervention. Their vocal changes across the month were quantified with (a) acoustic measures on fundamental frequency (fo), vocal intensity, jitter and shimmer, harmonics-to-noise ratio, and smoothed cepstral peak prominence and (b) Voice Handicap Index (VHI-10) and Vocal Fatigue Index (VFI) scores. RESULTS: Analysis of covariance showed significantly larger changes (significant decreases) in conversational fo and in jitter for the experimental group relative to the control group. Post hoc pairwise comparisons following repeated-measures analysis of variance showed significant decreases in conversational fo and in jitter across the month for the experimental group. No significant differences in VHI-10 and VFI scores were found between the groups. CONCLUSIONS: Vocal demand-related changes in acoustic measures could be partially mitigated with the vocal hygiene program. Future studies with a more refined intervention program and more long-term follow-up are recommended to better understand the long-term benefits of vocal hygiene programs on primary school teachers.

Associations between Oral Human Herpesvirus-6 and -7 and Periodontal Conditions in Older Adults.

BACKGROUND: The associations between oral human herpesvirus-6 (HHV-6) and HHV-7, periodontal conditions, and lifestyle-related diseases, such as hypertension, diabetes, and dyslipidemia, have not been fully investigated in older adults. METHODS: Seventy-four older patients who visited Hiroshima University Hospital were enrolled. Tongue swab samples were employed, and a real-time polymerase chain reaction was performed to detect HHV-6 and HHV-7 DNA. Dental plaque accumulation, probing pocket depth, and bleeding on probing (BOP) (i.e., a sign of periodontal inflammation) were examined. The periodontal inflamed surface area (PISA) value (i.e., an indicator of the severity of periodontitis) was also examined. RESULTS: Of the 74 participants, one participant (1.4%) was HHV-6 DNA-positive and 36 participants (48.6%) were HHV-7 DNA-positive. A significant association between HHV-7 DNA and probing depth was found (p = 0.04). The HHV-7 DNA-positive participants had a higher positive rate of a ≥6-mm periodontal pocket with BOP (25.0%) than the HHV-7 DNA-negative participants (7.9%). Additionally, the HHV-7 DNA-positive participants had a higher PISA value than the HHV-7 DNA-negative participants. However, there was no significant association between HHV-7 and the PISA value (p = 0.82). No significant association was found between HHV-7 and lifestyle-related diseases (p > 0.05). CONCLUSIONS: Oral HHV-7 infection is associated with a deep periodontal pocket.

The Reliability and Validity of the Mandarin Chinese Version of the Vocal Fatigue Index: Preliminary Validation.

PURPOSE: This study attempted to develop and to preliminarily validate the Mandarin Chinese version of the Vocal Fatigue Index (VFI) as a standardized self-assessment questionnaire tool for potential clinical applications. METHOD: The experimental procedure involved (a) cross-cultural adaptation of the VFI into the Mandarin Chinese version (CVFI), (b) evaluation by an expert panel, (c) back translation, (d) pilot testing, and (e) validation of the questionnaire by three participant groups: 50 with voice disorders, 50 occupational voice users (at-risk group), and 50 with normal voice (control group). Internal consistency, test-retest reliability, content validity, and convergent validity of the CVFI were examined, and discriminatory ability (diagnostic accuracy) for distinguishing between the groups was evaluated. RESULTS: Results showed high internal consistency (Cronbach's alpha ≥ .8817 for the total CVFI scores for all groups), high test-retest reliability (intraclass correlation coefficients ≥ .9072, p < .001 for the total CVFI scores for all groups), high content validity (total content validity index = 0.9368), and high convergent validity (Pearson r ≥ .8155, p < .001 between the total CVFI scores and Factors 1 and 2 scores). Significant differences between the three groups were found in all scores. Receiver operating characteristic analysis revealed a high diagnostic accuracy for distinguishing between the disorders group and the normal group (area under the curve ≥ 0.927, p < .001 for the total CVFI scores and Factors 1 and 2 scores), with cutoff scores of ≥ 36 (total CVFI score), ≥ 23.5 (Factor 1 score), ≥ 7.5 (Factor 2 score), and ≤ 6.5 (Factor 3 score). CONCLUSIONS: These findings suggested that the CVFI could be a reliable and valid self-assessment tool for the clinical evaluation of vocal fatigue in Mandarin Chinese-speaking populations. A full-scale validation study of the CVFI is recommended to verify these results.

Development of a Chemical Cocktail That Rescues Mouse Brain Demyelination in a Cuprizone-Induced Model.

Oligodendrocytes are glial cells located in the central nervous system (CNS) that play essential roles in the transmission of nerve signals and in the neuroprotection of myelinated neurons. The dysfunction or loss of oligodendrocytes leads to demyelinating diseases such as multiple sclerosis (MS). To treat demyelinating diseases, the development of a therapy that promotes remyelination is required. In the present study, we established an in vitro method to convert human fibroblasts into induced oligodendrocyte-like cells (iOLCs) in 3 days. The induced cells displayed morphologies and molecular signatures similar to oligodendrocytes after treatment with valproic acid and exposure to the small molecules Y27632, SU9516, and forskolin (FSK). To pursue the development of a cell-free remyelination therapy in vivo, we used a cuprizone-induced demyelinated mouse model. The small molecules (Y27632, SU9516, and FSK) were directly injected into the demyelinated corpus callosum of the mouse brain. This combination of small molecules rescued the demyelination phenotype within two weeks as observed by light and electron microscopy. These results provide a foundation for exploring the development of a treatment for demyelinating diseases via regenerative medicine.

Novel Tumor-Specific Antigens for Immunotherapy Identified From Multi-omics Profiling in Thymic Carcinomas.

Thymic carcinoma (TC) is the most aggressive thymic epithelial neoplasm. TC patients with microsatellite instability, whole-genome doubling, or alternative tumor-specific antigens from gene fusion are most likely to benefit from immunotherapies. However, due to the rarity of this disease, how to prioritize the putative biomarkers and what constitutes an optimal treatment regimen remains largely unknown. Therefore, we integrated genomic and transcriptomic analyses from TC patients and revealed that frameshift indels in KMT2C and CYLD frequently produce neoantigens. Moreover, a median of 3 fusion-derived neoantigens was predicted across affected patients, especially the CATSPERB-TC2N neoantigens that were recurrently predicted in TC patients. Lastly, potentially actionable alterations with early levels of evidence were uncovered and could be used for designing clinical trials. In summary, this study shed light on our understanding of tumorigenesis and presented new avenues for molecular characterization and immunotherapy in TC.

MutScape: an analytical toolkit for probing the mutational landscape in cancer genomics.

Cancer genomics has been evolving rapidly, fueled by the emergence of numerous studies and public databases through next-generation sequencing technologies. However, the downstream programs used to preprocess and analyze data on somatic mutations are scattered in different tools, most of which require specific input formats. Here, we developed a user-friendly Python toolkit, MutScape, which provides a comprehensive pipeline of filtering, combination, transformation, analysis and visualization for researchers, to easily explore the cohort-based mutational characterization for studying cancer genomics when obtaining somatic mutation data. MutScape not only can preprocess millions of mutation records in a few minutes, but also offers various analyses simultaneously, including driver gene detection, mutational signature, large-scale alteration identification and actionable biomarker annotation. Furthermore, MutScape supports somatic variant data in both variant call format and mutation annotation format, and leverages caller combination strategies to quickly eliminate false positives. With only two simple commands, robust results and publication-quality images are generated automatically. Herein, we demonstrate the ability of MutScape to correctly reproduce known results using breast cancer samples from The Cancer Genome Atlas. More significantly, discovery of novel results in cancer genomic studies is enabled through the advanced features in MutScape. MutScape is freely available on GitHub, at https://github.com/anitalu724/MutScape.

Differential whole-genome doubling and homologous recombination deficiencies across breast cancer subtypes from the Taiwanese population.

Whole-genome doubling (WGD) is an early macro-evolutionary event in tumorigenesis, involving the doubling of an entire chromosome complement. However, its impact on breast cancer subtypes remains unclear. Here, we performed a comprehensive and quantitative analysis of WGD and its influence on breast cancer subtypes in patients from Taiwan and consequently highlight the genomic association between WGD and homologous recombination deficiency (HRD). A higher manifestation of WGD was reported in triple-negative breast cancer, conferring high chromosomal instability (CIN), while HER2 + tumors exhibited early WGD events, with widely varied CIN levels, compared to luminal-type tumors. An association of higher activity of de novo indel signature 2 with WGD and HRD in Taiwanese breast cancer patients was reported. A control test between WGD and pseudo non-WGD samples was further employed to support this finding. The study provides a better comprehension of tumorigenesis in breast cancer subtypes, thus assisting in personalized treatment.

CNVIntegrate: the first multi-ethnic database for identifying copy number variations associated with cancer.

Human copy number variations (CNVs) and copy number alterations (CNAs) are DNA segments (>1000 base pairs) of duplications or deletions with respect to the reference genome, potentially causing genomic imbalance leading to diseases such as cancer. CNVs further cause genetic diversity in healthy populations and are predominant drivers of gene/genome evolution. Initiatives have been taken by the research community to establish large-scale databases to comprehensively characterize CNVs in humans. Exome Aggregation Consortium (ExAC) is one such endeavor that catalogs CNVs, of nearly 60 000 healthy individuals across five demographic clusters. Furthermore, large projects such as the Catalogue of Somatic Mutations in Cancer (COSMIC) and the Cancer Cell Line Encyclopedia (CCLE) combine CNA data from cancer-affected individuals and large panels of human cancer cell lines, respectively. However, we lack a structured and comprehensive CNV/CNA resource including both healthy individuals and cancer patients across large populations. CNVIntegrate is the first web-based system that hosts CNV and CNA data from both healthy populations and cancer patients, respectively, and concomitantly provides statistical comparisons between copy number frequencies of multiple ethnic populations. It further includes, for the first time, well-cataloged CNV and CNA data from Taiwanese healthy individuals and Taiwan Breast Cancer data, respectively, along with imported resources from ExAC, COSMIC and CCLE. CNVIntegrate offers a CNV/CNA-data hub for structured information retrieval for clinicians and scientists towards important drug discoveries and precision treatments. Database URL: http://cnvintegrate.cgm.ntu.edu.tw/.

Evolutionary Trajectories and Genomic Divergence in Localized Breast Cancers after Ipsilateral Breast Tumor Recurrence.

The evolutionary trajectories that drive clinical and therapeutic consequences in localized breast cancers (BCs) with ipsilateral breast tumor relapse (IBTR) remain largely unknown. Analyses of longitudinal paired whole-exome sequencing data from 10 localized BC patients with IBTR reveal that, compared to primary breast tumors, homologous recombination (HR) deficiency, inactivation of the HR pathway, chromosomal instability, and somatic driver mutations are more frequent. Furthermore, three major models of evolution in IBTR are summarized, through which relative contributions of mutational signatures shift, and the subclonal diversity expansions are shown. Optimal treatment regimens are suggested by the clinically relevant molecular features, such as HR deficiency (20%) or specific alterations (30%) with sensitivity to available FDA-approved drugs. Finally, a rationale for the development of the therapeutic management framework is provided. This study sheds light on the complicated evolution patterns in IBTR and has significant clinical implications for future improvement of treatment decisions.

A Mandarin Chinese Reading Passage for Eliciting Significant Vocal Range Variations.

Purpose There is a lack of standardized Mandarin reading text material that could potentially elicit significant variations in fundamental frequency (F0) and in vocal intensity for clinical voice evaluation. In this study, a phonetically balanced "Three Bears Passage" was developed based on the classical "Goldilocks" story for this purpose. The hypothesis was that the vocal range (F0 range and Intensity range) elicited while reading aloud the passage could be similar to that of the voice range profile (VRP) obtained with sustained vowel production and significantly different from that while reading an existing standard Mandarin passage. Method One hundred twenty young adults (60 men and 60 women, aged 25.22 ± 3.74 years) without vocal pathologies and vocal training participated in the study. Vocal range data, including F0-related measures and intensity-related measures, were obtained with Praat in order to construct VRPs for the sustained /a/ and to construct speech range profiles for passage reading. Results F0 range and intensity range for the new passage were significantly larger than those for the existing Mandarin passage for both male and female participants. In particular, the F0 range for the passage was not significantly different from that of the VRP for male participants. Conclusions These findings suggested that the new passage could allow individuals without vocal training to demonstrate a large vocal range in both F0 and vocal intensity, in a passage reading setting in a relatively short time. Further studies should be pursued with the passage to investigate the vocal range and vocal potential of individuals with voice disorders.

Effect of Training With Anchors on Auditory-Perceptual Evaluation of Dysphonia in Speech-Language Pathology Students.

Purpose Auditory-perceptual evaluation is essential for the clinical assessment of voice disorders. Unstable perceptual voice evaluation has been shown for inexperienced listeners as compared to expert listeners. We examined the effects of perceptual training with external auditory anchors with and without immediate feedback on the evaluation of roughness and breathiness of natural, nonsynthesized speech stimuli (reading of a standard passage) in speech-language pathology students. Method Perceptual voice evaluation and training with anchors using a visual analog scale was implemented with a computer software. Forty-eight speech-language pathology students were randomly assigned into three groups, feedback group (Group F), no feedback group (Group NF), and control group (Group C), attending one training session and four assessment sessions (before training, immediately after training, and 1 and 7 weeks after training). Group F received training with anchors with immediate feedback, Group NF received training without immediate feedback, and Group C received sham training (exposure session). Results Training with anchors significantly increased the rating accuracy (agreement with expert ratings) on both roughness and breathiness for Group F, with the effects lasting for 7 weeks. No significant changes in rating accuracy with training were observed for Group NF and Group C. No improvements in intra- and interrater reliability as well as intrarater agreement were observed in all three groups, whereas interrater agreement on breathiness (but not roughness) significantly increased for all groups, with the effect lasting for 7 weeks only for Group F. Conclusions These findings suggested that perceptual training with external auditory anchors and the use of immediate feedback could be effective for facilitating the development of perceptual voice evaluation skills in speech-language pathology students. Further studies involving more extensive training with stimuli covering a full range of dysphonia severity categories and improvements in design of the training protocol are recommended to verify these results.

Phonation Threshold Pressure/Flow for Reflecting Glottal Closure in Unilateral Vocal Fold Paralysis.

OBJECTIVE: Previous theoretical analysis predicted that phonation threshold flow (PTF) could be a more sensitive aerodynamic measure than phonation threshold pressure (PTP) for reflecting glottal incompetence. This study investigated the feasibility of whether PTP and PTF may differentiate subjects with unilateral adductor vocal fold paralysis and paresis (UAVFP) from those without, and whether PTP and PTF could reflect the extent of incomplete glottal closure associated with UAVFP. METHODS: PTP and PTF were quantified for 13 subjects with UAVFP and 21 control subjects with normal voice, and the normalized glottal gap area (NGGA) based on videostroboscopic image analysis was quantified for subjects with UAVFP. RESULTS: Significant differences in both PTP and PTF were found between subjects with UAVFP and control subjects. Receiver operating characteristic analysis indicated a higher discriminatory ability of PTP for differentiating subjects with UAVFP from those without (area under the curve of 0.905 for PTP, 0.678 for PTF), yet a significant positive correlation was found between PTF and NGGA (Spearman's ρ = 0.571) but not between PTP and NGGA (ρ = -0.364). CONCLUSION: Results supported the feasibility of using PTP and PTF as potential diagnostic indicators for reflecting glottal closure in UAVFP, with PTP potentially more sensitive for differentiating subjects with and without incomplete glottal closure. These preliminary findings were limited by the small sample size, with further studies needed to verify whether PTF could be more sensitive for reflecting the extent of incomplete glottal closure, as predicted theoretically. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E1598-E1604, 2021.

4-Acetylantroquinonol B Inhibits Osteoclastogenesis by Inhibiting the Autophagy Pathway in a Simulated Microgravity Model.

Astronauts suffer from 1-2% bone loss per month during space missions. Targeting osteoclast differentiation has been regarded as a promising strategy to prevent osteoporosis in microgravity (µXg). 4-acetylantroquinonol B (4-AAQB), a ubiquinone from Antrodia cinnamomea, has shown anti-inflammatory and anti-hepatoma activities. However, the effect of 4-AAQB on µXg-induced osteoclastogenesis remains unclear. In this study, we aimed to explore the mechanistic impact of 4-AAQB on osteoclast formation under µXg conditions. The monocyte/macrophage-like cell line RAW264.7 was exposed to simulated µXg (Rotary Cell Culture System; Synthecon, Houston, TX, USA) for 24 h and then treated with 4-AAQB or alendronate (ALN) and osteoclast differentiation factor receptor activator of nuclear factor kappa-B ligand (RANKL). Osteoclastogenesis, bone resorption activity, and osteoclast differentiation-related signaling pathways were analyzed using tartrate-resistant acid phosphatase (TRAP) staining, actin ring fluorescent staining, bone resorption, and western blotting assays. Based on the results of TRAP staining, actin ring staining, and bone resorption assays, we found that 4-AAQB significantly inhibited µXg-induced osteoclast differentiation. The critical regulators of osteoclast differentiation, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, and dendritic cell-specific transmembrane protein (DC-STAMP), were consistently decreased. Meanwhile, osteoclast apoptosis and cell cycle arrest were also observed along with autophagy suppression. Interestingly, the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) showed similar effects to 4-AAQB. In conclusion, we suggest that 4-AAQB may serve as a potential agent against µXg-induced osteoclast formation.

Effects of a 6-Week Straw Phonation in Water Exercise Program on the Aging Voice.

Purpose Semi-occluded vocal tract (SOVT) exercises with tubes or straws have been widely used for a variety of voice disorders. Yet, the effects of longer periods of SOVT exercises (lasting for weeks) on the aging voice are not well understood. This study investigated the effects of a 6-week straw phonation in water (SPW) exercise program. Method Thirty-seven elderly subjects with self-perceived voice problems were assigned into two groups: (a) SPW exercises with six weekly sessions and home practice (experimental group) and (b) vocal hygiene education (control group). Before and after intervention (2 weeks after the completion of the exercise program), acoustic analysis, auditory-perceptual evaluation, and self-assessment of vocal impairment were conducted. Results Analysis of covariance revealed significant differences between the two groups in smoothed cepstral peak prominence measures, harmonics-to-noise ratio, the auditory-perceptual parameter of breathiness, and Voice Handicap Index-10 scores postintervention. No significant differences between the two groups were found for other measures. Conclusions Our results supported the positive effects of SOVT exercises for the aging voice, with a 6-week SPW exercise program being a clinical option. Future studies should involve long-term follow-up and additional outcome measures to better understand the efficacy of SOVT exercises, particularly SPW exercises, for the aging voice.

Nucleocapsid phosphorylation and RNA helicase DDX1 recruitment enables coronavirus transition from discontinuous to continuous transcription.

Coronaviruses contain a positive-sense single-stranded genomic (g) RNA, which encodes nonstructural proteins. Several subgenomic mRNAs (sgmRNAs) encoding structural proteins are generated by template switching from the body transcription regulatory sequence (TRS) to the leader TRS. The process preferentially generates shorter sgmRNA. Appropriate readthrough of body TRSs is required to produce longer sgmRNAs and full-length gRNA. We find that phosphorylation of the viral nucleocapsid (N) by host glycogen synthase kinase-3 (GSK-3) is required for template switching. GSK-3 inhibition selectively reduces the generation of gRNA and longer sgmRNAs, but not shorter sgmRNAs. N phosphorylation allows recruitment of the RNA helicase DDX1 to the phosphorylated-N-containing complex, which facilitates template readthrough and enables longer sgmRNA synthesis. DDX1 knockdown or loss of helicase activity markedly reduces the levels of longer sgmRNAs. Thus, coronaviruses employ a unique strategy for the transition from discontinuous to continuous transcription to ensure balanced sgmRNAs and full-length gRNA synthesis.

A recombinant peroxisome proliferator response element-driven luciferase assay for evaluation of potential environmental obesogens.

A recombinant Huh7-PPRE-Luc cell line for analyzing the peroxisome proliferator response element (PPRE)-driven luciferase activity was established. The cells exhibited a good dose-response induction in PPRE-driven luciferase activity by three subtypes of peroxisome proliferator-activated receptor (PPAR) agonists as well as by a retinoid X receptor agonist, 9-cis-retinoic acid. Among five environmental chemicals tested, benzyl butyl phthalate and bisphenol induced PPRE-driven luciferase activation in Huh7-PPRE-Luc cells and caused adipogenic differentiation of 3T3-L1 cells. This recombinant Huh7-PPRE-Luc cell line would be useful for screening potential environmental obesogens with PPAR activity.

Emissions of polychlorinated dibenzo-p-dioxins and dibenzofurans from a heavy oil-fueled power plant in northern Taiwan.

We measured the concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) from the flue gas and the ambient atmosphere of a power plant fueled by heavy oil in northern Taiwan. The mean emission concentration and I-TEQ concentration of total PCDD/Fs were 0.292 ng/Nm(3) and 0.016 ng I-TEQ/Nm(3), respectively. All PCDD/F emission concentrations in the flue gas were supposed to meet the Environmental Protection Administration Executive Yuan, R.O.C. standard (1.0 ng I-TEQ/Nm(3) from 2008). Furthermore, the mean I-TEQ concentration in the ambient atmosphere was 0.011 pg I-TEQ/Nm(3), which was much lower than the environmental quality standards for dioxins in Japan (0.6 pg TEQ/Nm(3)). Also, the PCDD/F emission factor was 0.188 ng I-TEQ/L fuel, which was comparable to the data issued in US EPA [EPA, Locating and estimating air emissions from sources of dioxins and furans, Office of Air Quality Planning and Standards, Research Triangle Park, NC, DCN No. 95-298130-54-01, 1997] (0.2 ng I-TEQ/L of fuel). Also, the result of the correlations of PCDD/Fs and operational parameters illustrated that the positively significant correlation (r=0.502, p=0.048) was found only between PCDD/Fs (I-TEQ) and the flue gas emission temperature (125-157 degrees C). However, PCDD-TEQ/PCDF-TEQ ratios were statistically significantly associated with the decreased flue gas flow (r=-0.659, p=0.006), moisture (r=-0.612, p=0.012) and flue gas temperature (r=-0.503, p=0.047). For proper environmental management of dioxins, it is necessary to establish a complete emission inventory of PCDD/Fs, and, in particular, the government should pay more attention to power plants to address the information shortage.

Glycogen synthase kinase-3 regulates the phosphorylation of severe acute respiratory syndrome coronavirus nucleocapsid protein and viral replication.

Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication.

Differential effect of arecoline on the endogenous dioxin-responsive cytochrome P450 1A1 and on a stably transfected dioxin-responsive element-driven reporter in human hepatoma cells.

Dioxin-responsive element-mediated chemical activated luciferase expression (DRE-CALUX) is one of alternative bioassays for the determination of dioxin levels. We have previously established a DRE-CALUX cell line, Huh7-DRE-Luc, by using stable transfection of Huh-7 cells with a reporter plasmid (4xDRE-TATA-Luc) carrying a DRE-driven firefly luciferase gene. It was also shown that arecoline, a major areca nut alkaloid, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cytochrome P450 1A1 (CYP1A1) activation in Huh-7 cells. The TCDD-activated aryl hydrocarbon receptor (AhR) induces the DRE-CALUX activation and CYP1A1 gene expression via binding to DRE in promoter regions of these dioxin-responsive genes. In the present study, the effect of arecoline on the TCDD-induced activation of DRE-CALUX and CYP1A1 enzyme in Huh7-DRE-Luc and Huh-7 cells, respectively, was examined. It was found that arecoline inhibited TCDD-induced CYP1A1 activation and however enhanced TCDD-induced DRE-CALUX activation. This finding indicates the differential effect of arecoline on the endogenous dioxin-responsive CYP1A1 and on a stably transfected DRE-driven reporter in human hepatoma cells. The present study suggests that induction of DRE-CALUX alone does not necessarily parallel with endogenous CYP1A1 gene expression, and that the reporter assay may detect interactions that are not functional in endogenous gene.