A pragmatic feasibility trial of the Primary Care Intervention for PTSD: A health service delivery model to reduce health disparities for low-income and BIPOC youth.

OBJECTIVE: This study is a non-randomized pragmatic trial to assess the feasibility and acceptability of the Primary Care Intervention for Posttraumatic stress disorder (PCIP) (Srivastava et al., 2021), an Integrated Behavioral Health Care treatment for PTSD in adolescents. METHOD: Following routine clinic procedures, youth who were suspected of having trauma-related mental health symptoms were referred by their primary care providers to integrated care social workers for evaluation. The integrated care social workers referred the first 23 youth whom they suspected of having PTSD to the research study. Twenty youth consented to the study and 19 completed the pre-assessment (17 female; mean age = 19.32, SD = 2.11; range 14-22 years). More than 40% identified as Black and a third as Hispanic/Latinx. PCIP mechanisms and clinical outcomes were assessed pre- and post-treatment, and at one-month follow-up. Participants and therapists completed post-treatment qualitative interviews to assess feasibility and acceptability, and treatment sessions were audio recorded to assess fidelity. RESULTS: Findings suggest high acceptability, satisfaction, and feasibility of the PCIP delivered in "real-life" safety net pediatric primary care. Integrated care social workers had high treatment fidelity. Despite the small sample size, there was significant improvement in symptom scores of anxiety (g = 0.68, p = 0.02) and substance use (g = 0.36, p = 0.04) from pre to post, and depression symptoms (g = 0.38, p = 0.04) from pre to follow-up. Qualitative data from patients who completed exit interviews and integrated social workers indicated high satisfaction with the treatment, with some participants reporting that the integrated intervention was more acceptable and less stigmatizing than seeking mental health care outside of primary care. CONCLUSIONS: The PCIP may improve treatment engagement and access for vulnerable youth. Promising findings of high acceptability, feasibility, and initial clinical effectiveness suggest that PCIP warrants larger-scale study as part of routine care in pediatric integrated care.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

The SARS-CoV-2 accessory protein Orf3a is not an ion channel, but does interact with trafficking proteins.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as a viroporin. Here we show that neither SARS-CoV-2 nor SARS-CoV-1 form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a basic aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion.

Knowledge, attitudes, and practices of veterinary professionals towards ticks and tick-borne diseases in Illinois.

Objective: A lack of standardized surveillance or reporting of tick-borne diseases (TBDs) in Illinois creates uncertainty for veterinarians regarding TBDs occurring within their practice geography or which TBDs may be encroaching on their area from neighboring territories. Therefore, the objective of this study was to gauge the knowledge, attitudes, and practices of veterinary professionals in Southern and Central Illinois to establish a foundation for targeting educational and outreach programs that address knowledge gaps. Sample: 72 veterinary professionals in Central and Southern Illinois. Procedures: An online knowledge, attitudes, and practices survey was distributed to veterinary professionals in Southern and Central Illinois. Poisson regression analyses were conducted to determine factors associated with knowledge scores and the estimated number of TBD cases diagnosed. Results: Knowledge scores were significantly higher among veterinary practitioners with recent (within the last 5 years) training on TBD. The number of cases of TBD diagnosed was higher among those reporting concern about TBD, and among those who routinely test for TBDs. The types of diseases diagnosed were heavily influenced by the diagnostic method used. Clinical relevance: This study paints a cohesive picture of human factors associated with diagnosing veterinary diseases and TBD prevalence in Southern and Central Illinois. Our results highlight the importance and practical value of veterinary continuing education on ticks and TBDs for both companion animals and public health. Building capacity for training veterinarians in parasitology using partnerships between academia and industry may strengthen the knowledge and understanding of ticks and tick-borne pathogens in the veterinary community.

Development of an Illustrated Scale for Acute Radiation Dermatitis in Breast Cancer Patients.

PURPOSE: Scales for rating acute radiation dermatitis (ARD) have not been validated despite decades of clinical use, and little is known regarding the relationship between toxicity scores and patient-reported symptoms. Skin tone also complicates assessment of ARD, and as such we sought to design an illustrated scale to consistently describe ARD across several skin tone types in breast cancer patients undergoing radiation (RT). METHODS AND MATERIALS: Patients undergoing RT for breast cancer were enrolled on a prospective study with photographs obtained at 2-week intervals. Photographs were clustered according to the apparent severity of acute radiation dermatitis and a descriptive photonumeric scale was developed. Four clinically experienced raters used both the illustrated photonumeric scale and the Common Terminology Criteria for Adverse Events to independently score the collection of photographs in 2 independent sessions. RESULTS: Among 80 unique patients with 192 photographs, 47 patients (59%) completed questionnaires about their symptoms during RT. Physicians completed toxicity forms at the point-of-care for 52 patients (65%). Photonumeric ratings compared against patient reports of dry and moist desquamation demonstrated high specificity (95% and 93%, respectively) and negative predictive value (84% and 92%), indicating correct identification of patients who did not report dry or moist desquamation. The sensitivity and positive predictive value for separate measures of dry and moist desquamation were considerably lower. A combined measure of any desquamation (dry or moist) portrayed higher diagnostic accuracy, resulting in 72% sensitivity, 93% specificity, 75% positive predictive value, and 92% negative predictive value. Photonumeric ratings of dry or moist desquamation were significantly associated with patient reports of itching, burning or stinging, hurting, and swelling. CONCLUSIONS: The Michigan scale for acute radiation dermatitis is a simple grading rubric that is distinguished by characterization of its intra- and interrater reliability and diagnostic accuracy, correlation with patient-reported symptoms of bother and pain, and applicability across the spectrum of skin pigmentation.

Prevalence and staging of non-alcoholic fatty liver disease among patients with heart failure with preserved ejection fraction.

Insulin resistance and altered energy metabolism is common in non-alcoholic fatty liver disease (NAFLD) and appears to also be associated with myocardial dysfunction. We aimed to evaluate prevalence, staging and clinical features correlated with NAFLD among patients with heart failure with preserved ejection fraction (HFpEF). Adults with HFpEF were prospectively enrolled. Demographic and clinical data were collected. NAFLD was defined based on liver biopsy, abdominal imaging or ICD-coding and the absence of other liver diseases. Descriptive, bivariate and multivariable analyses were performed. 181 patients were analyzed. The median age was 70 with 89% white, 59% female, median BMI 35.1, and 48% with diabetes. NAFLD was present in 27% of the full cohort and 50% of those with imaging. In patients with imaging, multivariable analysis identified diabetes (OR 3.38, 95% CI 1.29-8.88) and BMI (OR 1.11, 95% CI 1.04-1.19) as independent correlates of NAFLD. 54% of NAFLD patients had a NAFLD fibrosis score consistent with advanced fibrosis. Cirrhosis was present in 6.6% of patients overall and 11.5% with imaging. NAFLD patients had a higher frequency of advanced heart failure (75% vs 55%, p 0.01). NAFLD has a two-fold higher prevalence in HFpEF compared to the general population and is independently associated with BMI and diabetes. Patients with HFpEF and NAFLD also appeared to have more advanced fibrosis including cirrhosis suggesting a potential synergistic effect of cardiac dysfunction on fibrosis risk in NAFLD. This data supports consideration for evaluation of underlying liver disease in HFpEF patients.

Assessing Barriers to Healthy Eating in Hospitalized Older Adults With Heart Failure: Psychometric Properties of Two Questionnaires.

BACKGROUND: Dietary sodium excess and malnutrition have been associated with poor outcomes in heart failure (HF). Few previous studies have examined the barriers to following a low-sodium, nutritionally robust diet in hospitalized patients with HF. METHODS AND RESULTS: As part of a dietary intervention pilot study, 76 inpatients with HF (age 71 ±â€¯8 years, 30% female, 30% black, 36% Hispanic/Latino) completed 2 questionnaires, the Dietary Sodium Restriction Questionnaire (DSRQ) and the Brief Dietary Psychosocial Scale (BDPS), to assess challenges in following a low-sodium, nutritionally complete diet. We assessed the factor structure of the DSRQ and BDPS with confirmatory and exploratory factor analysis (CFA and EFA). CFA did not support the established 3-factor solution for the DSRQ; instead, EFA indicated that a 2-factor solution (subjective norms/attitudes and perceived behavioral control) provided the best fit for the data. EFA supported 4 separate factors for the BDPS, as in its original derivation. Cronbach's alphas supported internal consistency reliability for both scales (DSRQ: 0.85-0.94; BDPS: 0.72-0.95). CONCLUSIONS: In a mixed-ethnicity group of hospitalized older patients with HF, the DSRQ and BDPS have reasonable psychometric properties. These questionnaires may help identify barriers to healthy dietary practices and facilitate nutritional interventions in this high-risk population.

Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin.

Bestrophin (BEST1-4) ligand-gated chloride (Cl-) channels are activated by calcium (Ca2+). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel's principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca2+ binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.

Distinct regions that control ion selectivity and calcium-dependent activation in the bestrophin ion channel.

Cytoplasmic calcium (Ca2+) activates the bestrophin anion channel, allowing chloride ions to flow down their electrochemical gradient. Mutations in bestrophin 1 (BEST1) cause macular degenerative disorders. Previously, we determined an X-ray structure of chicken BEST1 that revealed the architecture of the channel. Here, we present electrophysiological studies of purified wild-type and mutant BEST1 channels and an X-ray structure of a Ca2+-independent mutant. From these experiments, we identify regions of BEST1 responsible for Ca2+ activation and ion selectivity. A "Ca2+ clasp" within the channel's intracellular region acts as a sensor of cytoplasmic Ca2+. Alanine substitutions within a hydrophobic "neck" of the pore, which widen it, cause the channel to be constitutively active, irrespective of Ca2+. We conclude that the primary function of the neck is as a "gate" that controls chloride permeation in a Ca2+-dependent manner. In contrast to what others have proposed, we find that the neck is not a major contributor to the channel's ion selectivity. We find that mutation of a cytosolic "aperture" of the pore does not perturb the Ca2+ dependence of the channel or its preference for anions over cations, but its mutation dramatically alters relative permeabilities among anions. The data suggest that the aperture functions as a size-selective filter that permits the passage of small entities such as partially dehydrated chloride ions while excluding larger molecules such as amino acids. Thus, unlike ion channels that have a single "selectivity filter," in bestrophin, distinct regions of the pore govern anion-vs.-cation selectivity and the relative permeabilities among anions.

Crystal structure of the human two-pore domain potassium channel K2P1.

Two-pore domain potassium (K(+)) channels (K2P channels) control the negative resting potential of eukaryotic cells and regulate cell excitability by conducting K(+) ions across the plasma membrane. Here, we present the 3.4 angstrom resolution crystal structure of a human K2P channel, K2P1 (TWIK-1). Unlike other K(+) channel structures, K2P1 is dimeric. An extracellular cap domain located above the selectivity filter forms an ion pathway in which K(+) ions flow through side portals. Openings within the transmembrane region expose the pore to the lipid bilayer and are filled with electron density attributable to alkyl chains. An interfacial helix appears structurally poised to affect gating. The structure lays a foundation to further investigate how K2P channels are regulated by diverse stimuli.

Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways.

TGF-beta and BMP receptor kinases activate Smad transcription factors by C-terminal phosphorylation. We have identified a subsequent agonist-induced phosphorylation that plays a central dual role in Smad transcriptional activation and turnover. As receptor-activated Smads form transcriptional complexes, they are phosphorylated at an interdomain linker region by CDK8 and CDK9, which are components of transcriptional mediator and elongation complexes. These phosphorylations promote Smad transcriptional action, which in the case of Smad1 is mediated by the recruitment of YAP to the phosphorylated linker sites. An effector of the highly conserved Hippo organ size control pathway, YAP supports Smad1-dependent transcription and is required for BMP suppression of neural differentiation of mouse embryonic stem cells. The phosphorylated linker is ultimately recognized by specific ubiquitin ligases, leading to proteasome-mediated turnover of activated Smad proteins. Thus, nuclear CDK8/9 drive a cycle of Smad utilization and disposal that is an integral part of canonical BMP and TGF-beta pathways.

Alcohol regulates gene expression in neurons via activation of heat shock factor 1.

Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10-60 mM), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.