Using Affinity Pulldown Assays to Study Protein-Protein Interactions of Human NEIL1 Glycosylase and the Checkpoint Protein RAD9-RAD1-HUS1 (9-1-1) Complex.

Affinity pulldown is a powerful technique to discover novel interaction partners and verify a predicted physical association between two or more proteins. Pulldown assays capture a target protein fused with an affinity tag and analyze the complexed proteins. Here, we detail methods of pulldown assays for two high-affinity peptide fusion tags, Flag tag (DYKDDDDK) and hexahistidine tag (6xHis), to study protein-protein interactions of human NEIL1 glycosylase and the checkpoint protein complex RAD9-RAD1-HUS1 (9-1-1). We uncover unique interactions between 9-1-1 and NEIL1, which suggest a possible inhibitory role of the disordered, phosphorylated C-terminal region of RAD9 in regulating NEIL1 activity in base excision repair through lack of physical association of 9-1-1 and NEIL1.

Outcomes of a Live Messaging, Blended Care Coaching Program Among Adults With Symptoms of Anxiety: Pragmatic Retrospective Cohort Study.

BACKGROUND: Anxiety disorders are common and can be debilitating. In addition, various barriers exist that can hinder access to adequate care. Coaching that is grounded in evidence-based interventions and delivered via synchronous (ie, live) text-based messaging could potentially increase the reach of mental health services among populations who select this modality instead of other services (eg, face-to-face coaching and psychotherapy). In addition, the delivery of live messaging coaching within a blended care model has the potential to combine the benefits of coaching with those of evidence-based digital mental health tools. OBJECTIVE: This real-world study evaluates the anxiety and satisfaction outcomes of live messaging coaching blended with digital tools (ie, digital exercises and activities). METHODS: This was a retrospective cohort study of 121 adults with moderate levels of anxiety symptoms at the beginning of coaching (Generalized Anxiety Disorder-7 [GAD-7] scores: range 8-14). Participants received an employer-offered blended messaging coaching (BMC) program, and those who opted to receive all live coaching sessions via text-based messaging were included. Anxiety symptom severity was regularly measured by using the GAD-7 scale. Using growth curve models, the change in GAD-7 scores over the course of BMC was evaluated, as were the effects of text-based coaching sessions on GAD-7 scores. The proportion of participants that had a reliable improvement in anxiety symptom severity (GAD-7 score reduction of ≥4) or subclinical symptom severity (GAD-7 score of <8) at the end of care was also estimated. Participants also self-reported their likelihood of recommending their live messaging coach to someone with similar needs. RESULTS: At baseline, the average GAD-7 score was 9.88 (SD 1.80). Anxiety symptom severity significantly decreased with each week in the BMC program (week: b=-1.04; P<.001), and the rate of decline in anxiety symptom severity decreased over time (week2: b=0.06; P<.001). Each live messaging coaching session was associated with significantly lower anxiety symptom severity during the week of the coaching session (b=-1.56; P<.001) and the week immediately following the session (b=-1.03; P<.001). Overall, 86% (104/121) of participants had subclinical symptom severity or a reliable reduction in anxiety symptom severity by the end of care. Further, 33.1% (40/121) of participants reported coaching satisfaction levels; of the 40 participants in this subset, 37 (92.5%) were likely or extremely likely to recommend their live messaging coach. CONCLUSIONS: BMC that provides coaching sessions via live messaging can be beneficial for adults with moderate symptoms of anxiety who qualify for and self-select this care modality. Large-scale studies with longer follow-ups are needed.

Blended Care Therapy for Depression and Anxiety: Outcomes across Diverse Racial and Ethnic Groups.

BACKGROUND: Studies have reported positive outcomes of blended care therapy (BCT), which combines face-to-face care with internet modules. However, there is insufficient evidence of its effectiveness across racial and ethnic groups. This study evaluated outcomes of a BCT program, which combined video psychotherapy with internet cognitive-behavioral modules, across race and ethnicity. METHODS: Participants were 6492 adults, with elevated anxiety (Generalized Anxiety Disorder-7 [GAD-7] ≥ 8) and/or depression (Patient Health Questionnaire-9 [PHQ-9] ≥ 10) symptoms, enrolled in employer-offered BCT. Changes in anxiety (GAD-7) and depression (PHQ-9) symptoms during treatment were evaluated using individual growth curve models. Interaction terms of time with race and ethnicity tested for between-group differences. Treatment satisfaction was assessed using a Net Promoter measure (range = 1 (lowest satisfaction) to 5 (greatest satisfaction)). RESULTS: Participants' self-reported race and ethnicity included Asian or Pacific Islander (27.5%), Black or African American (5.4%), Hispanic or Latino (9.3%), and White (47.2%). Anxiety symptoms decreased during treatment (p < 0.01), with greater reductions among Hispanic or Latino participants compared to White participants (p < 0.05). Depressive symptoms decreased across treatment (p < 0.01), with significantly greater decreases among some racial and ethnic groups compared to White participants. Declines in anxiety and depressive symptoms slowed across treatment (p's < 0.01), with statistically significant differences in slowing rates of depressive symptoms across some racial and ethnic groups. Among participants with responses (28.45%), average treatment satisfaction ranged from 4.46 (SD = 0.73) to 4.67 (SD = 0.68) across race and ethnicity (p = 0.001). Racial and ethnic differences in outcomes were small in magnitude. CONCLUSIONS: BCT for anxiety and depression can be effective across diverse racial and ethnic groups.

Real-World Evaluation of a Large-Scale Blended Care-Cognitive Behavioral Therapy Program for Symptoms of Anxiety and Depression.

Introduction: Prior studies have supported the effectiveness of blended interventions for anxiety and depression; however, outcomes research of large-scale blended interventions for these conditions is limited. Objective: To investigate the outcomes of scaled-up blended care (BC) cognitive behavioral therapy (CBT), a program that combined video-based psychotherapy with internet CBT, for symptoms of anxiety and depression. Materials and Methods: Participants were 6,738 U.S.-based adults with elevated symptoms of anxiety (Generalized Anxiety Disorder-7 [GAD-7] ≥ 8) and/or depression (Patient Health Questionnaire-9 [PHQ-9] ≥ 10) at baseline who received BC-CBT as an employer-offered mental health benefit. The primary outcomes, anxiety (GAD-7) and depression (PHQ-9) symptoms, were routinely measured in the program. Recovery and reliable improvement in outcomes were calculated, and growth curve models evaluated change in outcomes during treatment and the effects of engaging in psychotherapy sessions on outcomes. Results: On average, participants received treatment for 7.6 (standard deviation = 6.2) weeks. By the end of care, 5,491 (81.5%) participants had reliable improvement in either anxiety or depression symptoms; in addition, 5,535 (82.1%) fell below the clinical threshold for either anxiety or depression symptoms (i.e., recovered). Declines in anxiety and depression symptoms were statistically significant over the course of BC-CBT (both p's < 0.01), with the rate of decline significantly decreasing for each outcome as treatment progressed (both p's < 0.01). Each psychotherapy session completed was significantly associated with lower anxiety and depression symptoms during the week of the session and the subsequent week (all p's < 0.01). Conclusions: This real-world study provides evidence that scaled-up BC-CBT can be effective in the treatment of symptoms of anxiety and depression.

Parathyroid Hormone Senses Extracellular Calcium To Modulate Endocrine Signaling upon Binding to the Family B GPCR Parathyroid Hormone 1 Receptor.

Parathyroid hormone (PTH) binds to a family B G protein coupled receptor, parathyroid hormone 1 receptor (PTH1R). One of its functions is to regulate Ca2+ homeostasis in bone remodeling, during which Ca2+ can reach up to 40 mM. A truncated version of PTH, PTH(1-34), can fully activate PTH1R and has been used for osteoporosis treatments. Here, we used fluorescence anisotropy to examine the binding of PTH(1-34) to PTH1R purified in nanodiscs (PTH1R-ND) and found that the affinity increases 5-fold in the presence of 15 mM Ca2+. However, PTHrP(1-36), another truncated endogenous agonist for PTH1R, does not show this Ca2+ effect. Mutations of Glu19 and Glu22 in PTH(1-34) that are not conserved in PTHrP(1-36) largely abolished the Ca2+ effect. The results support that PTH(1-34) not only activates PTH1R but also uniquely senses Ca2+. This dual function of a peptide hormone is a novel observation that couples changes in extracellular environment with endocrine signaling. Understanding this can potentially reveal the complex role of PTH signaling in bone remodeling and improve the PTH(1-34) treatment for osteoporosis.

ß-Peptide bundles: Design. Build. Analyze. Biosynthesize.

Peptides containing ß-amino acids are unique non-natural polymers known to assemble into protein-like tertiary and quaternary structures. When composed solely of ß-amino acids, the structures formed, defined assemblies of 14-helices called ß-peptide bundles, fold cooperatively in water solvent into unique and discrete quaternary assemblies that are highly thermostable, bind complex substrates and metal ion cofactors, and, in certain cases, catalyze chemical reactions. In this Perspective, we recount the design and elaboration of ß-peptide bundles and provide an outlook on recent, unexpected discoveries that could influence research on ß-peptides and ß-peptide bundles (and ß-amino acid-containing proteins) for decades to come.

Design and high-resolution structure of a ß³-peptide bundle catalyst.

Despite the widespread exploration of α-peptides as catalysts, there are few examples of ß-peptides that alter the course of a chemical transformation. Our previous work demonstrated that a special class of ß(3)-peptides spontaneously self-assembles in water into discrete protein-like bundles possessing unique quaternary structures and exceptional thermodynamic stability. Here we describe a series of ß(3)-peptide bundles capable of both substrate binding and chemical catalysis--ester hydrolysis. A combination of kinetic and high-resolution structural analysis suggests an active site triad composed of residues from at least two strands of the octameric bundle structure.

Relationship between side-chain branching and stoichiometry in ß(3)-peptide bundles.

The stability and stoichiometry of ß(3)-peptide bundles is influenced by side-chain identity. ß(3)-peptides containing ß(3)-homoleucine on one helical face assemble into octamers, whereas those containing ß(3)-homovaline form tetramers. From a structural perspective, the side chains of ß(3)-homoleucine and ß(3)-homovaline differ in terms of both side-chain length and γ-carbon branching. To evaluate the extent to which these two parameters control ß(3)-peptide bundle stoichiometry, we synthesized the ß(3)-peptide Acid-3Y, which contains ß(3)-homoisoleucine in place of ß(3)-homoleucine or ß(3)-homovaline. Acid-3Y assembles into a stable tetramer whose stability resembles that of the previously characterized Acid-VY tetramer. These results suggest that ß(3)-peptide bundle stoichiometry is dominated by the presence or absence of γ-carbon branching on core side chains.