Nanobody-Mediated Dualsteric Engagement of the Angiotensin Receptor Broadens Biased Ligand Pharmacology.

Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or ß-arrestin (TRV027). While the nanobody itself acts as a probe-specific neutral or negative allosteric ligand of N-terminally 6e-tagged AT1R, nanobody conjugation to orthosteric ligands had varying effects on Gq dissociation and ß-arrestin plasma membrane recruitment. The potency of certain AngII analogs was enhanced up to 100-fold, and some conjugates behaved as partial agonists, with up to a 5-fold decrease in maximal efficacy. Nanobody conjugation also biased the signaling of TRV055 and TRV056 toward Gq, suggesting that Gq bias at AT1R can be modulated through molecular mechanisms distinct from those previously elucidated. Both competition radioligand binding experiments and functional assays demonstrated that orthosteric antagonists (angiotensin receptor blockers) act as non-competitive inhibitors of all these nanobody-peptide conjugates. This proof-of-principle study illustrates the array of pharmacological patterns that can be achieved by incorporating neutral or negative allosteric pharmacophores into dualsteric ligands. Nanobodies directed toward linear epitopes could provide a rich source of allosteric reagents for this purpose. SIGNIFICANCE STATEMENT: Here we engineer bitopic (dualsteric) ligands for epitope-tagged angiotensin II type 1 receptor by conjugating angiotensin II or its biased analogs to an epitope-specific nanobody (antibody fragment). Our data demonstrate that nanobody-mediated interactions with the receptor N-terminus endow angiotensin analogs with properties of allosteric modulators and provide a novel mechanism to increase the potency, modulate the maximal effect, or alter the bias of ligands.

Increased Actin Binding Is a Shared Molecular Consequence of Numerous SCA5 Mutations in ß-III-Spectrin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein ß-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the ß-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

Expanding the Trauma-Informed Care Measurement Toolkit: An Evaluation of the Attitudes Related to Trauma-Informed Care (ARTIC-45) Scale with SUD Workers in PIMH.

Human service organizations (HSO) have increasingly recognized the value of employing trauma-informed care (TIC) in a variety of practice settings. Evidence suggests that effectively adopting TIC has shown client improvements. Organizational barriers to TIC implementation, however, exist. To improve TIC practice, the attitudes related to trauma-informed care (ARTIC) scale was developed to measure staff attitudes and beliefs towards TIC. The ARTIC has been widely adopted by researchers without evaluating its psychometric performance in diverse practice settings. The purpose of this study was to independently validate the ARTIC scale drawn from a sample of staff (n = 373) who provide services to substance-using parents. Psychometric tests were conducted to evaluate how the ARTIC performs with our HSO population. Results from a confirmatory factor analysis showed poor fit (X2 = 2761.62, df = 2.96; RMSEA = 0.07 [0.07, 0.08]; CFI = 0.72). An exploratory factor analysis was conducted to analyze how the data fit with our specific population, yielding 10 factors. Finally, a qualitative inter-item analysis of these factors was conducted, resulting in nine factors. Our findings suggest that measuring TIC attitudes and beliefs may vary according to field of practice and ethno-racially diverse workers. Further refinement of the ARTIC may be necessary for various services domains.

Increased actin binding is a shared molecular consequence of numerous spinocerebellar ataxia mutations in ß-III-spectrin.

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein ß-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the ß-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with early age of symptom onset. Altogether, the data indicate increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

Early-phase drug discovery of ß-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

ß-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein ß-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant ß-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P ß-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for ß-III-spectrin ABD modulators.

Harsh parenting among veterans: parents' military-related PTSD, mentalization, and pre-military trauma.

Objectives: Veteran parents experiencing posttraumatic stress disorder (PTSD) may resort to harsh parenting. The indirect pathway from parental military-related PTSD to harsh parenting, and the moderating role of parents' pre-military trauma histories, has been less explored. Informed by mentalization theory, as well as trauma-sensitive and posttraumatic growth perspectives, we aim to explore the associations between veteran parents' military-related PTSD, mentalization, harsh parenting, and prior trauma before military service. Methods: Data were collected from an online research panel of 509 veteran parents with children under 10. We employed Structural Equation Models to test indirect and moderating effects. Results: We identified an indirect effect of parental pre-mentalization from military PTSD to harsh parenting [corporal punishment: b = 0.35, p < 0.001, 95% CI (0.23, 0.46); psychological aggression: b = 0.14, p < 0.001, 95% CI (0.09, 0.19)]. Multi-group analysis on four parent groups (parents with only pre-military physical trauma, parents with only pre-military psychological trauma, parents with both pre-military physical and psychological trauma, and parents with no pre-military physical or psychological trauma) highlighted differences in these associations, particularly between parents with only pre-military physical trauma and those without any physical and psychological trauma. The military-related PTSD effects on psychological aggression, corporal punishment, and pre-mentalization were all significantly higher for parents without pre-military physical and psychological trauma. Conclusion: Modifying parents' interpretation of their child's mental states can potentially counteract the effects of veterans' military PTSD on harsh parenting. Family-based programs should be created considering veteran parents' pre-military trauma histories.

Religion, politics and COVID-19 risk perception among urban residents in Malawi.

INTRODUCTION: Majority of Malawians have not yet adopted COVID-19 mitigation measures despite having knowledge about its infectivity, morbidity, and fatality. Understanding drivers of hesitancy to adoption of COVID-19 mitigation measures is critical as it can inform prevention programs. This study explores Malawians' COVID-19 risk perception, and the associated constraints in the adoption of mitigation efforts. A Health Belief Model (HBM) approach was used to understand perceived factors that undermine public health COVID-19 messages to reduce the spread of the pandemic in Malawi. METHODS: The study applied rapid appraisal and photovoice qualitative inquiry to comprehend risk perception regarding COVID-19. We purposively selected 52 participants from three major cities in Malawi. Audio and video interviews were transcribed verbatim, and transcripts were coded manually to derive key themes and concepts. RESULTS: The study identified that social factors particularly religious and political beliefs influenced COVID-19 risk perception. Specific religious beliefs pertaining to individuals recognizing signs of the 'Christian apocalypse' were particularly associated with lower risk perceptions. Politically, participants believed COVID-19 lockdown measures were a ploy by the then-ruling party to remain in power. CONCLUSION: The study suggests that religious beliefs and political environment undermine self -perceived risk of contracting COVID-19 among urban dwellers in Malawi. We recommend that diverse actors in Malawi should collaborate to promote the dissemination of accurate COVID-19 discourses and reduce the severity of the pandemic's impact in Malawi.

LuNER: Multiplexed SARS-CoV-2 detection in clinical swab and wastewater samples.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2-3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

Launching a saliva-based SARS-CoV-2 surveillance testing program on a university campus.

Regular surveillance testing of asymptomatic individuals for SARS-CoV-2 has been center to SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants' experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Robotic RNA extraction for SARS-CoV-2 surveillance using saliva samples.

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI-FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against the gold standard, nasopharyngeal swab specimens. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.

IGI-LuNER: single-well multiplexed RT-qPCR test for SARS-CoV-2.

Commonly used RT-qPCR-based SARS-CoV-2 diagnostics require 2-3 separate reactions or rely on detection of a single viral target, adding time and cost or risk of false-negative results. Currently, no test combines detection of widely used SARS-CoV-2 E- and N-gene targets and a sample control in a single, multiplexed reaction. We developed the IGI-LuNER RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (NER). This combined, cost-effective test can be performed in 384-well plates with detection sensitivity suitable for clinical reporting, and will aid in future sample pooling efforts, thus improving throughput of SARS-CoV-2 detection.

Integrating social science and genetics: news from the political front.

There has been growing interest in the use of genetic models to expand the understanding of political preferences, attitudes, and behaviors. Researchers in the social sciences have begun incorporating these models and have revealed that genetic differences account for individual differences in political beliefs, behaviors, and responses to the political environment. The first Integrating Genetics and the Social Sciences Conference, held at Boulder, Colorado in May of 2010, brought together these researchers. As a result, we jointly review the last 5 years of research in this area. In doing so, we explicate the methods, findings, and limitations of behavior genetic approaches, including twin designs, association studies, and genome-wide analyses, in their application toward exploring political preferences.

Treatment of adults with attention-deficit/hyperactivity disorder.

This review focuses on the treatment of attention deficit hyperactivity disorder (ADHD) in adults. It briefly addresses prevalence, diagnostic and differential diagnostic issues specific to adults. Stimulant medication, non-stimulant medication, and psychosocial treatments are thoroughly reviewed. For each class of medication possible mechanism of action, efficacy and side effects are summarized. Special attention is given to the pharmacological treatment for patients with adult ADHD and various comorbidities. In summary, stimulant medications are most effective and combined medication and psychosocial treatment is the most beneficial treatment option for most adult patients with ADHD.

Treatment of adults with attention-deficit/hyperactivity disorder.

This review focuses on the treatment of attention deficit hyperactivity disorder (ADHD) in adults. It briefly addresses prevalence, diagnostic and differential diagnostic issues specific to adults. Stimulant medication, non-stimulant medication, and psychosocial treatments are thoroughly reviewed. For each class of medication possible mechanism of action, efficacy and side effects are summarized. Special attention is given to the pharmacological treatment for patients with adult ADHD and various comorbidities. In summary, stimulant medications are most effective and combined medication and psychosocial treatment is the most beneficial treatment option for most adult patients with ADHD.

Current issues and trends in the diagnosis and treatment of adults with ADHD.

Attention-deficit/hyperactivity disorder (ADHD) has been commonly thought of as a childhood disorder that diminished over time. It is one of the most common developmental disorders and it is estimated that ADHD affects 5-10% of children. Two-thirds of children with ADHD will continue to have symptoms of ADHD that persist throughout adolescence. Longitudinal studies have demonstrated that symptoms of ADHD can also remain in adulthood, affecting 4.4% of the adult population. However, diagnosing adults with ADHD can prove difficult because they often find that their symptoms are egosyntonic. In addition, the development of comorbid conditions, such as anxiety, depression, personality disorders or substance abuse, can often overshadow underlying ADHD symptoms. Nonetheless, treatments such as stimulant and nonstimulant medication (e.g., atomoxetine), and cognitive-behavior therapy have been effective in treating adults with ADHD. This article reviews the prevalence of adults with ADHD, followed by a discussion of the neurobiological and genetic underpinnings of the disorder. Issues regarding the diagnosis and treatment of ADHD are also addressed.