HAP40 is a conserved central regulator of Huntingtin and a potential modulator of Huntington's disease pathogenesis.

Perturbation of huntingtin (HTT)'s physiological function is one postulated pathogenic factor in Huntington's disease (HD). However, little is known how HTT is regulated in vivo. In a proteomic study, we isolated a novel ~40kDa protein as a strong binding partner of Drosophila HTT and demonstrated it was the functional ortholog of HAP40, an HTT associated protein shown recently to modulate HTT's conformation but with unclear physiological and pathologic roles. We showed that in both flies and human cells, HAP40 maintained conserved physical and functional interactions with HTT. Additionally, loss of HAP40 resulted in similar phenotypes as HTT knockout. More strikingly, HAP40 strongly affected HTT's stability, as depletion of HAP40 significantly reduced the levels of endogenous HTT protein while HAP40 overexpression markedly extended its half-life. Conversely, in the absence of HTT, the majority of HAP40 protein were degraded, likely through the proteasome. Further, the affinity between HTT and HAP40 was not significantly affected by polyglutamine expansion in HTT, and contrary to an early report, there were no abnormal accumulations of endogenous HAP40 protein in HD cells from mouse HD models or human patients. Lastly, when tested in Drosophila models of HD, HAP40 partially modulated the neurodegeneration induced by full-length mutant HTT while showed no apparent effect on the toxicity of mutant HTT exon 1 fragment. Together, our study uncovers a conserved mechanism governing the stability and in vivo functions of HTT and demonstrates that HAP40 is a central and positive regulator of endogenous HTT. Further, our results support that mutant HTT is toxic regardless of the presence of its partner HAP40, and implicate HAP40 as a potential modulator of HD pathogenesis through its multiplex effect on HTT's function, stability and the potency of mutant HTT's toxicity.

Factors associated with symptom severity in Canadian youth with mental health and/or addictions concerns accessing service navigation.

This study investigated the factors associated with clinical symptoms and level of functioning at baseline and after 4 months of navigation, in youth with mental health and/or addiction concerns involved with a family navigation service. Participants in this pre-post study were caregivers who accessed a mental health and addictions navigation service between March 2018 and July 2019 on behalf of their youth aged 13-26 who had mental health and/or addiction concerns. Evaluations were conducted at baseline and at 4 months after entering navigation. The dependent variable, youth mental health symptoms and functioning, was assessed by caregivers using the Symptoms and Functioning Severity Scale. The sample included 345 caregivers of youth (youth: mean age 18 years, 57% male). At intake, using multiple linear regression, caregiver strain (ß = 0.481, p < 0.001), youth completion of ADLs (ß = -0.156, p = 0.002) and youth motivation (ß = -0.110, p = 0.021) contributed significantly to variance in symptoms and functioning (R2  = 0.373, p < 0.001). Using ANOVA to compare complete pre/post data (n = 115), there was a significant main effect of time on the youth symptoms and functioning score (F[1, 107] = 9.278, p = 0.003). Furthermore, the interaction of time, youth completion of ADLs (low vs. high at baseline) and youth motivation (low vs. high at baseline) was significant (F[1, 107] = 4.699, p = 0.032). Youth with low motivation and low completion of ADLs at baseline experienced the largest improvements in symptoms and functioning after 4 months of navigation. These findings suggest that this group of youth should be a primary target in mental health and addiction navigation.

Pregnancy and childbirth during incarceration: A qualitative systematic review of lived experiences.

BACKGROUND: Incarcerated individuals who experience pregnancy or childbirth in correctional facilities face unique considerations for obstetric care and consequently are at greater risk of adverse maternal and fetal outcomes. OBJECTIVES: To characterise patient experiences regarding pregnancy and childbirth during incarceration via qualitative synthesis. SEARCH STRATEGY: Medline-OVID, EMBASE, CINAHL, Sociological Abstracts, Social Work Abstracts, Web of Science, Scopus and PsycInfo were systematically searched from inception to 24 December 2020. Supplementary searches were performed using the Scopus database. SELECTION CRITERIA: Only original, peer-reviewed literature was examined. Eligible studies were assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Qualitative Research. RESULTS: After screening 4173 original database citations, 24 studies that met inclusion criteria were included and analysed via thematic analysis. The 24 studies included perspectives from 645 female patients who had experienced incarceration, 69 healthcare providers and 70 prison staff. Key patient-reported concerns for the well-being of pregnant individuals during incarceration included mental health challenges, dehumanisation of prenatal care and delivery, lack of privacy, stigma, psychological trauma, lack of emotional support and shackle usage during pregnancy and/or labour. The studies reported a lack of support for patients to access female correctional officers or guards, privacy during intimate examinations, timely medical care and support for breastfeeding. Above all, the psychological trauma of separation from one's newborn after birth was of utmost devastation. CONCLUSIONS: Our systematic review highlights the dire need for accountability and interventions to improve pregnancy and childbirth care for incarcerated individuals. TWEETABLE ABSTRACT: This systematic review describes lived experiences of pregnancy & childbirth during incarceration, including dehumanisation, psychological trauma, and use of shackles.

Evaluated interventions addressing developmental transitions for youth with mental health disorders: a meta-analysis.

PURPOSE: The objective of this meta-analysis was to provide a quantitative synthesis of the effects of studies evaluating developmentally appropriate programs or interventions for transition-age youth with mental health disorders. METHODS: Studies, between January 1992 and March 2021, were included if they contained a sample population with a median age between 12 and 25 years and with a mental health disorder and described the results of health interventions addressing aspects of developmental transitions. Independent reviewers screened study texts and assessed the risk of bias. Random effects meta-analysis was used to pool data on standardized mean differences. RESULTS: Under neurodevelopmental studies (6), the effect size of interventions measuring social outcomes was 1.00 (95% CI: -0.01 to 2.00), parental stress levels was -0.10 (95% CI:-0.74 to 0.55), autism symptoms was -0.40 (95% CI: -1.58 to 0.78), and self-determination was 0.16 (95% CI:-0.38 to 0.70). Under mental illness studies (3), the effect size of interventions measuring adolescent depressive symptoms was 0.48 (95% CI: 0.01 to 0.96) and parental depressive symptoms was 1.09 (95% CI: 0.20 to 1.97). CONCLUSIONS: There is no effect of interventions except on parental depressive symptoms under mental illness studies. Further research with comparable outcomes and assessments is needed.Implications for rehabilitation:Interventions for youth with mental health disorders should be developmentally appropriate and incorporate elements to assist youth in multiple aspects of their lives.The following approaches should be considered in interventions: skills training, prevocational/vocational guidance, a client-centered approach, and/or an ecological/experiential approach.Intervention researchers and practitioners should incorporate similar outcome assessment tools and measures in order to allow for valid comparisons between intervention effectiveness.

Examining Device-Assessed Physical Activity During the Transition Into Emerging Adulthood: Results From the MovingU Study.

PURPOSE: The purpose of the present study was to conduct the first longitudinal investigation using accelerometers to assess physical activity behavior change during individuals' acute transition out of high school. METHODS: Participants in the current investigation were a part of a prospective cohort study called the MovingU Study. Participants were 163 adolescents (Mage = 16.9 ± .5; n = 88 females) recruited at the beginning of their final year at high school, with follow-up at 6 and 18 months. Participants wore the ActiGraph Link for 7 days at each assessment period. RESULTS: Results from the mixed-effects models found no significant differences in moderate-to-vigorous physical activity or total physical activity between baseline and follow-up at 6 months (Estimates = 1.91 and -2.26, p's > .05), respectively; however, significant differences in both moderate-to-vigorous physical activity and total physical activity between baseline and follow-up at 18 months after the transition out of highschool (Estimate = -123.62 and -15.38, p's < .01). CONCLUSIONS: Current findings provide additional compelling data to show precipitous declines in physical activity as adolescents transition out of high school and into emerging adulthood. Continued efforts to maintain or increase physical activity during adolescence and the prevention of physical activity declines as they enter emerging adulthood could have important public health benefits.

A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties.

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

Mechanism of action and antiviral activity of benzimidazole-based allosteric inhibitors of the hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTP-binding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.

Characterization of resistance to non-obligate chain-terminating ribonucleoside analogs that inhibit hepatitis C virus replication in vitro.

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2'-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-alpha results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2'-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2'-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections.

In vitro selection and characterization of hepatitis C virus serine protease variants resistant to an active-site peptide inhibitor.

The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

Hepatitis C virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons.

For many years our knowledge on hepatitis C virus (HCV) replication has been based on in vitro experiments or transfection studies. Recently, the first reliable system for studying viral replication in tissue culture cells was developed. Taking advantage of this system, we examined in detail the localization of viral nonstructural (NS) proteins in cells containing functional replication complexes. By fractionation experiments and immunomicroscopy, we observed that all NS proteins were associated with the endoplasmic reticulum (ER) membranes, confirming the hypothesis that the ER is the site of membrane-associated HCV RNA replication. Interestingly, NS3 and NS4A were preferentially localized in endoplasmic reticulum cisternae surrounding mitochondria, suggesting additional subcellular compartment-related functions for these viral proteins. Furthermore, the immunoelectron microscopy revealed the loss of the organization and other morphological alterations of the ER (convoluted cisternae and paracrystalline structures), resembling alterations observed in liver biopsies of HCV-infected individuals and in flavivirus-infected cells.