Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Problematic Use of Internet Associates with Poor Quality of Life via Psychological Distress in Invididuals with ADHD.

Background: Problematic use of internet (PUI) may have negative impacts on psychological distress and quality of life (QoL). This situation might be more profound in people with attention-deficit/hyperactivity disorder (ADHD) due to poorer behavioral control and regulatory capacity. However, there is little evidence regarding mediated effects in the associations between PUI, psychological distress, and QoL in people with ADHD. Aims: To investigate mediating effects of psychological distress in the associations of problematic smartphone use (PSPU), problematic use of social media (PUSM), and problematic gaming (PG) with QoL in individuals with ADHD. Methods and Procedures: PUI behaviors of participants with ADHD (n = 99) were assessed using the Smartphone Application-Based Addiction Scale, Bergen Social Media Addiction Scale, and Internet Gaming Disorder-Short Form. Psychological distress was assessed using the Depression, Anxiety, Stress Scale and QoL using the Kid-KINDL. Outcomes and Results: Psychological distress mediated the associations between PUI and different domains of QoL, except for self-esteem QoL. There were also positively direct effects between PG and physical QoL, PUSM and friends' QoL, and PSPU and physical QoL. Conclusions and Implications: PUI may associate with poor QoL in people with ADHD via psychological distress. Programs on reducing PUI for people with ADHD are needed.

Unraveling blunt-end RNA binding and ATPase-driven translocation activities of the RIG-I family helicase LGP2.

The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5'-triphosphate (5'ppp), Cap0, or Cap1 RNA-end does not influence LGP2's RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5-10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3'-strand, suggesting a 3'-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.

Different autism measures targeting different severity levels in children with autism spectrum disorder.

The Childhood Autism Rating Scale™, Second Edition (CARS™-2) and Social Responsiveness Scale™, Second Edition (SRS™-2) are two measures for identifying autism symptoms. The CARS™-2 has two versions: Standard (CARS-ST) and High-Functioning (CARS-HF). To better understand their properties, this study aimed to investigate: (1) the associations among the CARS-ST, CARS-HF and the SRS™-2, and (2) the severity consistency between the CARS-ST and the CARS-HF. A sample of 125 children with autism spectrum disorder was recruited (mean age: 80.98 months, SD = 16.08). Based on Verbal Comprehension Index (VCI), children were divided into two groups: low severity level of autism spectrum disorder (LSL-ASD: VCI ≥ 80) and high severity level of autism spectrum disorder (HSL-ASD: VCI < 80). All children were evaluated with the CARS-ST and the SRS™-2, and the HF group, with the CARS-HF as well. In the LSL group, the CARS-ST and the CARS-HF had high correlation (r = 0.852, p < .001). Both versions had small to moderate correlations with the SRS™-2 (r = 0.130-0.491). In the HSL group, no significant correlations were found between the CARS-ST and SRS™-2 (p > .05). The CARS-HF and the CARS-ST had low severity consistency (Kappa = 0.376, p < .01). The CARS-ST and the CARS-HF had high correlations but low severity consistency. Different correlation patterns were found between the CARS™-2 and the SRS™-2 in the LSL and HSL groups. The results should help clinicians better understand the properties of the measures and choose appropriate measures when assessing autism symptoms.

Exploring mediational roles for self-stigma in associations between types of problematic use of internet and psychological distress in youth with ADHD.

BACKGROUND: Several studies have linked the problematic use of the Internet (PUI) to psychological distress. Youth with attention deficit hyperactivity disorder (ADHD) are considered a particular disadvantaged population with a high risk of developing PUI, psychological distress, and self-stigma. Nonetheless, the interrelationships of PUI, self-stigma, and psychological distress in adolescents with ADHD are not well understood. AIMS: This study investigated whether self-stigma mediates relationships between different forms of PUI, such as problematic gaming (PG), problematic social media use (PSMU), problematic smartphone use (PSPU), and psychological distress (i.e., depression, anxiety, and stress), in children with ADHD. METHODS AND PROCEDURES: We recruited 100 youth with ADHD (mean age=10.80 [SD=3.07] years; 84 boys) from psychiatric outpatient clinics in Taiwan. All participants were assessed for PUI (via Internet Gaming Disorder-Short Form for PG, Bergan Social Medica Addiction Scale for PSMU, and Smartphone Application-Based Addiction Scale for PSPU), self-stigma (via Self-Stigma Short-Scale), and psychological distress (via Depression, Anxiety, Stress Scale). OUTCOMES AND RESULTS: The results of path and bootstrapping analyses indicated that self-stigma mediated the associations between PSMU and PSPU, but not PG, and depression, anxiety, and stress. CONCLUSIONS AND IMPLICATIONS: This study expands the extant literature by revealing that self-stigma mediates the association between specific forms of PUI and psychological distress in adolescents with ADHD. Interventions aimed at reducing self-stigma and PUI, particularly PSMU and PSPU, may help decrease psychological distress among adolescents with ADHD.

Rasch Modeling and Differential Item Functioning of the Self-Stigma Scale-Short Version among People with Three Different Psychiatric Disorders.

Self-stigma is prevalent in individuals with psychiatric disorders and can profoundly affect people. A unified assessment with sound psychometric properties is needed for evaluating self-stigma across psychiatric conditions. The aim of this study was to examine the psychometric properties of the Self-Stigma Scale-Short version (SSS-S) using Rasch modeling. Six-hundred and twelve participants with substance use disorders (n = 319), attention-deficit/hyperactivity disorder (n = 100), and schizophrenia (n = 193) completed the SSS-S. Rasch results confirmed the unidimensionality of the nine items of the SSS-S. The four-point Likert scale of the SSS-S reflected monotonical increases along the self-stigma continuum. No ceiling or floor effects were detected. Among the three subdomains of the SSS-S, cognitive items appeared to be the most robustly endorsed, and behavioral items were the least endorsed. Two items in the SSS-S displayed differential item functioning across the three diagnoses. Additionally, SSS-S scores showed weak to moderate correlation with depression, anxiety, and stress scale scores. The SSS-S had overall satisfactory psychometric properties. Healthcare professionals may use this assessment to assess self-stigma in multiple psychiatric groups, and information gained may facilitate improved care.

Mangrove colonization on tidal flats causes straightened tidal channels and consequent changes in the hydrodynamic gradient and siltation potential.

A healthy mangrove ecosystem includes diverse landscape structures, such as tidal flats, tidal channels, and areas with circulating waters, in addition to mangrove stands. The complex structure of mangrove forests affects the hydrodynamics and sediment transport behaviour of tidal channels. Understanding the influence of the mangrove invasion of tidal flats on the pattern and stability of tidal channels is essential. In this study, two types of remote sensing images, Google Earth images and aerial photographs, were collected to analyze the relationship between mangrove colonization and changes in tidal channel patterns. After applying binary image processing, these two kinds of images show similar abilities to discriminate the locations, extents, and boundaries of mangroves and tidal channels. We found that the mangrove area was inversely proportional to the tidal channel sinuosity and width. The tidal channels exhibited a meandering pattern with a wider width before the mangroves invaded the tidal flats. After the expansion of the mangroves, the tidal channels gradually transformed into a straight shape with a narrower width. After the mangroves developed into forests, the tidal channels maintained a straight and stable pattern. Since mangroves promote siltation and increase the elevation of the surrounding mudflats, the habitat suitability for mangroves in the neighbouring tidal flat areas may vary. These processes may help expand mangrove habitats, thereby compressing the area of flats and changing the shape of tidal channels. Due to tidal current effects, the unit stream power of a straight tidal channel is approximately twice that of a meandering channel, indicating that straight tidal channels have a stronger anti-siltation capability. Our research also found that the tidal channels may return to a meandering pattern when mangroves are degraded or die and their area decreases. This study provides key evidence that mangroves affect tidal channel types and hydrodynamic characteristics, thus providing a useful reference for restoring and managing estuarine mangrove ecosystems.

Frontal Beta Activity in the Meta-Intention of Children With Attention Deficit Hyperactivity Disorder.

Children with attention deficit hyperactivity disorder (ADHD) have high theta and low beta activity in the frontal lobe. The higher the theta/beta ratio, the lower the level of central nervous system (CNS) cortical arousal. However, there is seldom evidence between electroencephalograms (EEGs) and the patient's intentionality to regulate the cortical activity of executive attention tasks. We investigated whether children with ADHD intended to improve their performance in executive attention tasks and whether that increased their brain activity. Fifty-one children with ADHD (ADHD) and 51 typical developing (TD) children were investigated using focused attention (FA) and search attention (SA) tasks and a simultaneous EEG. The children were then regrouped as faster (ADHD-F, TD-F) and slower (ADHD-S, TD-S) depending on reaction time (RT). Quantitative EEGs of frontal lobe theta and beta activity at frontal F3, F4, and Fz were used. Twenty-eight (54.9%) ADHD children were regrouped as ADHD-S and 14 (27.5%) as TD-S. The ADHD-S group, however, had poorer FA and SA performance than the other 3 groups did: fewer correct answers, more frequent impulsive and missing errors, and higher RT variations. There were no significant differences in theta activity, but the TD-S group had higher beta activity than the ADHD-S group did. We conclude that the ADHD-F and ADHD-S groups had different attention processes. beta activity did not increase in the ADHD-S group, and their executive attention performance in the FA and SA tests was poor. It seems ADHD-S had poor meta-intention function. The frontal beta activity might be a feasible training target of neurofeedback in ADHD-S patients.

Opioid agonist treatment reduces losses in quality of life and quality-adjusted life expectancy in heroin users: Evidence from real world data.

BACKGROUND: This study estimated the long-term changes of opioid agonist treatment (OAT) in quality of life (QOL) and quantified the quality-adjusted life years (QALY) from the loss of quality-adjusted life expectancy (QALE) in heroin users. METHODS: A total of 1283 heroin users stratified by OAT were linked to the National Mortality Registry for 8 years (2006-2014) to obtain survival functions, which were extrapolated to lifetime by applying a rolling extrapolation algorithm to survival ratio between the sub-cohorts and age- and sex-matched referents simulated from vital statistics of Taiwan. We performed cross-sectional measurement of EQ-5D on 349 participants, including those with a valid state of OAT or non-OAT plus newly recruited consecutive patients, during 2015-2017 for utility values, while the QOL of referents were abstracted from the 2009 National Health Interview Survey. The QALE was calculated by summing the products of the mean QOL and survival rate throughout life. The QALE difference between the cohort and corresponding referents was the loss-of-QALE. RESULTS: QOL of the OAT group was significantly better than that of the non-OAT group in every domain of the EQ-5D, which was quantified to be 0.23 for utility after controlling for other variables. After extrapolation to 70 years, the estimated QALE and loss-of-QALE were 17.8 and 18.2 QALY for OAT subjects, respectively, while those of the non-OAT group were 9.2 and 27.9 QALY. CONCLUSIONS: Receiving OAT could reduce QALE lost by 9.7 QALYs compared with non-OAT after accounting for QOL differences along time and different age and sex distributions.

Differential Proteomics Reveals Discrete Functions of Proteins Interacting with Hypo- versus Hyper-phosphorylated NS5A of the Hepatitis C Virus.

Protein phosphorylation is a reversible post-translational modification that regulates many biological processes in almost all living forms. In the case of the hepatitis C virus (HCV), the nonstructural protein 5A (NS5A) is believed to transit between hypo- and hyper-phosphorylated forms that interact with host proteins to execute different functions; however, little was known about the proteins that bind either form of NS5A. Here, we generated two high-quality antibodies specific to serine 235 nonphosphorylated hypo- vs serine 235 phosphorylated (pS235) hyper-phosphorylated form of NS5A and for the first time segregated these two forms of NS5A plus their interacting proteins for dimethyl-labeling based proteomics. We identified 629 proteins, of which 238 were quantified in three replicates. Bioinformatics showed 46 proteins that preferentially bind hypo-phosphorylated NS5A are involved in antiviral response and another 46 proteins that bind pS235 hyper-phosphorylated NS5A are involved in liver cancer progression. We further identified a DNA-dependent kinase (DNA-PK) that binds hypo-phosphorylated NS5A. Inhibition of DNA-PK with an inhibitor or via gene-specific knockdown significantly reduced S232 phosphorylation and NS5A hyper-phosphorylation. Because S232 phosphorylation initiates sequential S232/S235/S238 phosphorylation leading to NS5A hyper-phosphorylation, we identified a new protein kinase that regulates a delicate balance of NS5A between hypo- and hyper-phosphorylation states, respectively, involved in host antiviral responses and liver cancer progression.

Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A.

The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.

Age at Exposure to Parental Suicide and the Subsequent Risk of Suicide in Young People.

BACKGROUND: We investigated the age at exposure to parental suicide and the risk of subsequent suicide completion in young people. The impact of parental and offspring sex was also examined. METHOD: Using a cohort study design, we linked Taiwan's Birth Registry (1978-1997) with Taiwan's Death Registry (1985-2009) and identified 40,249 children who had experienced maternal suicide (n = 14,431), paternal suicide (n = 26,887), or the suicide of both parents (n = 281). Each exposed child was matched to 10 children of the same sex and birth year whose parents were still alive. This yielded a total of 398,081 children for our non-exposed cohort. A Cox proportional hazards model was used to compare the suicide risk of the exposed and non-exposed groups. RESULTS: Compared with the non-exposed group, offspring who were exposed to parental suicide were 3.91 times (95% confidence interval [CI] = 3.10-4.92 more likely to die by suicide after adjusting for baseline characteristics. The risk of suicide seemed to be lower in older male offspring (HR = 3.94, 95% CI = 2.57-6.06), but higher in older female offspring (HR = 5.30, 95% CI = 3.05-9.22). Stratified analyses based on parental sex revealed similar patterns as the combined analysis. LIMITATIONS: As only register--based data were used, we were not able to explore the impact of variables not contained in the data set, such as the role of mental illness. CONCLUSION: Our findings suggest a prominent elevation in the risk of suicide among offspring who lost their parents to suicide. The risk elevation differed according to the sex of the afflicted offspring as well as to their age at exposure.

Opioid users with comorbid hepatitis C spent more time in agonist therapy: A 6-year observational study in Taiwan.

BACKGROUND: Hepatitis C virus (HCV) infection is highly prevalent among opioid agonist therapy (OAT) patients, but little is known about long-term OAT use among this population. METHODS: Subjects diagnosed as opioid dependence were recruited from Mar. 2006 to Jul. 2008 in a psychiatry center in southern Taiwan with the OAT censored in 2012, and their socio-demographics, drug use characteristics, and markers of blood-borne infection were assessed at entry. Correlates with HCV infection and OAT retention were analyzed by multivariate logistic regression. Retention (OAT utilization) was defined as the in-treatment period of OAT during the 6-year observation period. RESULTS: A total of 983 patients (88.3% men) were included. The prevalences of HCV and HIV infection were 91.4% and 17.9%, respectively. The mean duration of OAT during the study period was 2.3±0.8years. Significant correlates with HCV infection were retention of at least three years in OAT (AOR: 4.24, 95%CI: 1.49-12.03), ever sharing injection equipment (AOR: 227.04, 95%CI: 57.22-900.87), not living with family (AOR: 5.54, 95%CI: 1.45-21.16), lower educational attainment (AOR: 2.10, 95%CI: 1.15-3.82) and previous drug offense (AOR: 6.35, 95%CI: 1.69-23.83). Significant correlates with retention were HCV infection (AOR: 2.53, 95%CI: 1.30-4.93) and divorced or separation in marriage (AOR: 0.65, 95%CI: 0.44-0.96). CONCLUSIONS: This six-year observational study revealed a better retention in OAT if opioid-dependent individuals had comorbid hepatitis C. This provided opportunities for OAT patients with HCV infection to obtain medical treatment while staying in an OAT program. Further research could explore the possibility of eradicating comorbid HCV infection among these long-term treatment cases.

Serine 235 Is the Primary NS5A Hyperphosphorylation Site Responsible for Hepatitis C Virus Replication.

The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) is a phosphoprotein with two phosphorylation states: hypo- and hyperphosphorylation. Genetic mutation studies have demonstrated a cluster of serine residues responsible for NS5A hyperphosphorylation and functions in viral replication and assembly; however, the phosphorylation levels and potential interactions among the serine residues are unclear. We used three specific antibodies to measure NS5A phosphorylation at S222, S235, and S238 that were identified in our previous proteomics study. In the HCV (J6/JFH-1)-infected Huh7.5.1 cells, S222 phosphorylation was barely detected, whereas S235 phosphorylation and S238 phosphorylation were always detected in parallel in time and intracellular spaces. S235A mutation eliminated S238 phosphorylation whereas S238A mutation did not affect S235 phosphorylation, indicating that S235 phosphorylation occurs independently of S238 phosphorylation while S238 phosphorylation depends on S235 phosphorylation. In line with this, immunoprecipitation coupled with immunoblotting showed that S235 phosphorylation existed alone without S238 phosphorylation, whereas S238 phosphorylation existed only when S235 was phosphorylated on the same NS5A molecule. S235-phosphorylated NS5A constituted the primary hyperphosphorylated NS5A species. S235A mutation blunted viral replication, whereas S238A mutation did not affect replication. We concluded that S235 is the primary NS5A hyperphosphorylation site required for HCV replication. S238 is likely phosphorylated by casein kinase Iα, which requires a priming phosphorylation at S235.IMPORTANCE It has been known for years that the hepatitis C virus nonstructural protein 5A (NS5A) undergoes transition between two phosphorylation states: hypo- and hyperphosphorylation. It is also known that a cluster of serine residues is responsible for NS5A hyperphosphorylation and functions; however, the primary serine residue responsible for NS5A hyperphosphorylation is not clear. Here, we show for the first time that serine 235-phosphorylated NS5A constitutes the primary hyperphosphorylated NS5A species required for viral replication. We also show that NS5A phosphorylation among the serine residues is interdependent and occurs in a directional manner, i.e., phosphorylation at serine 235 leads to phosphorylation at serine 238. Our data provide the first proof-of-principle evidence that NS5A undergoes a sequential phosphorylation cascade.

Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases.

Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.

Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein.

Estimation of life expectancy and the expected years of life lost among heroin users in the era of opioid substitution treatment (OST) in Taiwan.

BACKGROUND: Opioid substitution treatment (OST) has been implemented in Taiwan since 2006. We estimated the life expectancy (LE) and expected years of life lost (EYLL) in a cohort of heroin users stratified by OST for comparison. METHODS: A total of 1283 heroin users recruited from 2006 to 2008 were linked to the National Mortality Registry until the end of 2011. Among them, 983 received OST, while 300 did not. Kaplan-Meier estimation for survival was performed, and it was extrapolated to 50 years to obtain the LE using a semi-parametric method. We further estimated the EYLL for both cohorts by subtracting their life expectancies from the age- and sex-matched referents of the general population. Cause-specific standardized mortality ratios (SMRs) were calculated and compared with the national cohort to validate the representativeness of this sample. RESULTS: After extrapolation to 50 years of survival, the estimated average LE and EYLL were 27.4 and 10.6 for OST subjects, respectively, while those of the non-OST were 20.2 and 18.4 years. The all-cause mortality rates (per 1000 person-years) in the observational period for the OST and non-OST group were 15.5 and 23.9, respectively, representing a 7.5- and 10.2-fold SMR compared to the general population, indicating a high representativeness for our sample. But SMR of suicide mortality elevated 16.2 and 3.1 folds in OST and non-OST group, respectively. CONCLUSIONS: OST saves 7.8 EYLL more than non-OST after accounting for lead time bias. Effective suicide prevention programs could enhance its life-saving effect, especially among those co-morbid with depressive disorders.