Efficient Cloning of Inserts for Phage Display by Golden Gate Assembly.

Phage display enables the discovery of high-affinity binders. In phage display, one commonly uses traditional cloning methods to insert DNA into the coding region of one of the five capsid proteins. Here we describe the use of a new vector with kanamycin resistance and BsaI sites for the utilization of Golden Gate cloning into the N-terminus of mature protein III. We also describe the successful pentavalent display of six different inserts: the AviD-tag, the Z-domain of protein A, the Myc-tag, the ALFA nanobody, the BC2 nanobody, and the Flag-tag.

Construction of an Ultra-Large Phage Display Library by Kunkel Mutagenesis and Rolling Circle Amplification.

An important contributor to the successful generation of recombinant affinity reagents via phage display is a large and diverse library. We describe, herein, the application of Kunkel mutagenesis and rolling circle amplification (RCA) to the construction of a 1.1 × 1011 member library, with only 26 electroporations, and isolation of low- to sub-nanomolar monobodies to a number of protein targets, including human COP9 signalosome subunit 5 (COPS5), HIV-1 Rev. binding protein-like protein (HRBL), X-ray repair cross-complementing 5/6 (Ku70/80) heterodimer, the receptor-binding domain (RBD) of SARS-CoV-2, and transforming growth factor beta 1 (TGF-ß1).

Expanding the chemical diversity of M13 bacteriophage.

Bacteriophage M13 virions are very stable nanoparticles that can be modified by chemical and genetic methods. The capsid proteins can be functionalized in a variety of chemical reactions without loss of particle integrity. In addition, Genetic Code Expansion (GCE) permits the introduction of non-canonical amino acids (ncAAs) into displayed peptides and proteins. The incorporation of ncAAs into phage libraries has led to the discovery of high-affinity binders with low nanomolar dissociation constant (K D) values that can potentially serve as inhibitors. This article reviews how bioconjugation and the incorporation of ncAAs during translation have expanded the chemistry of peptides and proteins displayed by M13 virions for a variety of purposes.

Cost-effectiveness analysis of deep transcranial magnetic stimulation relative to evidence-based strategies for treatment-refractory obsessive-compulsive disorder.

OBJECTIVE: This study examined the cost-effectiveness of deep transcranial magnetic stimulation (dTMS) for treatment refractory obsessive-compulsive disorder (OCD) relative to other established treatment options, including antidepressant medication (ADM), ADM + antipsychotic augmentation, real-world cognitive-behavioral therapy (ADM + CBT Effectiveness), clinical trial CBT (ADM + CBT), intensive outpatient program (IOP), partial hospitalization program (PHP), and PHP to IOP stepdown. METHODS: A decision analytic model was developed to evaluate the cost-effectiveness of dTMS relative to other established treatment alternatives for adults (18-64 years old) with refractory OCD. Building on Gregory et al. (2018), the model was parameterized with probabilistic and deterministic parameters from the literature and an outcomes database to perform a Monte Carlo simulation of a hypothetical cohort of 100,000 adults with OCD to estimate costs, and incremental cost-effectiveness ratio (ICER) for dTMS relative to each treatment strategy. Encounters took place from 2012 to 2015. Data for dTMS were taken from a recent multisite study. RESULTS: Although dTMS fit between ADM and ADM + CBT in overall costs, ADM + CBT had the lowest ICER and thus would be chosen before dTMS. dTMS was determined to be more cost effective relative to PHP/IOP stepdown, PHP, and IOP. CONCLUSION: dTMS is cost-effective, along the treatment continuum from outpatient medication management and CBT to more intensive, facilities-based approaches, and may be an incremental strategy to employ when higher intensity strategies are either not available, not financially feasible, or whilst on extended waits for admission to these higher levels of care.

Comparing efficacy of telehealth to in-person mental health care in intensive-treatment-seeking adults.

The heightened acuity in anxiety and depressive symptoms catalyzed by the COVID-19 pandemic presents an urgent need for effective, feasible alternatives to in-person mental health treatment. While tele-mental healthcare has been investigated for practicability and accessibility, its efficacy as a successful mode for delivering high-quality, high-intensity treatment remains unclear. This study compares the clinical outcomes of a matched sample of patients in a private, nation-wide behavioral health treatment system who received in-person, intensive psychological treatment prior to the COVID-19 pandemic (N = 1,192) to the outcomes of a distinctive group of patients who received telehealth treatment during the pandemic (N = 1,192). Outcomes are measured with respect to depressive symptoms (Quick Inventory of Depressive Symptomatology-Self-Report; QIDS-SR) and quality of life (Quality of Life Enjoyment and Satisfaction Questionnaire; Q-LES-Q). There were no significant differences in admission score on either assessment comparing in-person and telehealth groups. Patients in the partial hospitalization level of care stayed longer when treatment was remote. Results suggest telehealth as a viable care alternative with no significant differences between in-person and telehealth groups in depressive symptom reduction, and significant increases in self-reported quality of life across both groups. Future research is needed to replicate these findings in other healthcare organizations in other geographical locations and diverse patient populations.

Algorithmic Prediction of Restraint and Seclusion in an Inpatient Child and Adolescent Psychiatric Population.

BACKGROUND: Restraint and seclusion in an inpatient child and adolescent psychiatric population adversely affects the overall value and safety of care. Due to adverse events, negative outcomes, and associated costs, inpatient psychiatric hospitals must strive to reduce and ultimately eliminate restraint and seclusion with innovative, data-driven approaches. AIM: To identify patterns of client characteristics that are associated with restraint and seclusion in an inpatient child and adolescent psychiatric population. METHOD: A machine learning application of fast-and-frugal tree modeling was used to analyze the sample. RESULTS: The need for restraint and seclusion were correctly predicted for 73% of clients at risk (sensitivity), and 76% of clients were correctly predicted as negative or low risk (specificity), for needing restraint and seclusion based on the following characteristics: having a disruptive mood dysregulation disorder and/or attention-deficit hyperactivity disorder diagnosis, being 12 years old or younger, and not having a depressive and/or bipolar disorder diagnosis. CONCLUSIONS: The client characteristics identified in the predictive algorithm should be reviewed on admission to recognize clients at risk for restraint and seclusion. For those at risk, interventions should be developed into an individualized client treatment plan to facilitate a proactive approach in preventing behavioral emergencies requiring restraint and seclusion.

OCD in the time of COVID-19: A global pandemic's impact on mental health patients and their treatment providers.

Individuals with obsessive-compulsive disorder (OCD) have evidenced resilience against large-scale crises, although emerging research on the impact of COVID-19 is mixed. Little is known about the impact of COVID-19 on mental health providers. Items from an instrument evaluating the impact of the September 11, 2001, terrorist attack were adapted to measure the impact of COVID-19 on emotions, cognitions, and behaviors. Using a sample of 65 patients with primary OCD diagnoses and OCD treatment providers in intensive programs for OCD and anxiety, the authors found that COVID-19 evidenced a less significant overall impact on patients than providers. Specifically, providers reported more significant impact on the amount of time spent worrying about COVID-19, taking additional cleaning and sanitization precautions, and time spent socializing with loved ones. Findings support previous literature indicating that individuals with OCD demonstrate resilience to large-scale crises, and offer insights into the specific struggles of providers who treat OCD.

Plug and play with recombinant antibody fragments.

In this issue of Cell Chemical Biology, Hentrich et al. (2021) describe the application of the SpyCatcher technology to antibody discovery and validation. Fab-SpyTag fusion proteins can be expressed in the periplasm of protease-deficient bacteria and coupled in a modular manner to a variety of SpyCatcher-tagged proteins for improved assay performance.

Phosphorylated Gß is a directional cue during yeast gradient tracking.

Budding yeast cells interpret shallow pheromone gradients from cells of the opposite mating type, polarize their growth toward the pheromone source, and fuse at the chemotropic growth site. We previously proposed a deterministic, gradient-sensing model that explains how yeast cells switch from the intrinsically positioned default polarity site (DS) to the gradient-aligned chemotropic site (CS) at the plasma membrane. Because phosphorylation of the mating-specific Gß subunit is thought to be important for this process, we developed a biosensor that bound to phosphorylated but not unphosphorylated Gß and monitored its spatiotemporal dynamics to test key predictions of our gradient-sensing model. In mating cells, the biosensor colocalized with both Gß and receptor reporters at the DS and then tracked with them to the CS. The biosensor concentrated on the leading side of the tracking Gß and receptor peaks and was the first to arrive and stop tracking at the CS. Our data showed that the concentrated localization of phosphorylated Gß correlated with the tracking direction and final position of the G protein and receptor, consistent with the idea that gradient-regulated phosphorylation and dephosphorylation of Gß contributes to gradient sensing. Cells expressing a nonphosphorylatable mutant form of Gß exhibited defects in gradient tracking, orientation toward mating partners, and mating efficiency.

FN3-based monobodies selective for the receptor binding domain of the SARS-CoV-2 spike protein.

A phage library displaying 1010 variants of the fibronectin type III (FN3) domain was affinity selected with the biotinylated form of the receptor binding domain (RBD, residues 319-541) of the SARS-CoV-2 virus spike protein. Nine binding FN3 variants (i.e. monobodies) were recovered, representing four different primary structures. Soluble forms of the monobodies bound to several different preparations of the RBD and the S1 spike subunit, with affinities ranging from 3 to 14 nM as measured by bio-layer interferometry. Three of the four monobodies bound selectively to the RBD of SARS-CoV-2, with the fourth monobody showing slight cross-reactivity to the RBD of SARS-CoV-1 virus. Examination of binding to the spike fragments and its trimeric form revealed that the monobodies recognise at least three overlapping epitopes on the RBD of SARS-CoV-2. While pairwise tests failed to identify a monobody pair that could bind simultaneously to the RBD, one monobody could simultaneously bind to the RBD with the ectodomain of the cellular receptor angiotensin converting enzyme 2 (ACE2). All four monobodies successfully bound the RBD after overexpression in Chinese hamster ovary (CHO) cells as fusions to the Fc domain of human IgG1.

The rates of co-occurring behavioural addictions in treatment-seeking individuals with obsessive-compulsive disorder: a preliminary report.

Objectives: To assess the rates of co-occurring putative 'behavioural addictions' in patients with obsessive-compulsive disorder (OCD).Methods: Twenty-three international centres specialising in the treatment of OCD were invited to participate in a survey of the rates of behavioural addictions and other relevant comorbidity within their samples.Results: Sixteen of 23 (69.6%) invited centres from 13 countries had sufficient data to participate in the survey. The use of validated diagnostic tools was discrepant, with most centres relying on a 'clinical diagnosis' to diagnose behavioural addictions. The final sample comprised of 6916 patients with a primary diagnosis of OCD. The reported rates of behavioural addictions were as follows: 8.7% for problematic internet use, 6.8% for compulsive sexual behaviour disorder, 6.4% for compulsive buying, 4.1% for gambling disorder and 3.4% for internet gaming disorder.Conclusions: Behavioural addictions should be better assessed for patients with OCD. The absence of diagnostic scales developed specifically for behavioural addictions and overlapping obsessive-compulsive phenomena such as compulsive checking of information on the internet may explain the relatively high rate of problematic internet use in this sample. The study encourages better efforts to assess and to conceptualise the relatedness of behavioural addictions to obsessive-compulsive 'spectrum' disorders.

Predictors of treatment outcome for youth receiving intensive residential treatment for obsessive-compulsive disorder (OCD).

Little is known about the predictors of outcome from intensive residential treatment of OCD. This study aimed to examine age, gender, and baseline OCD severity, as well as measures of comorbid anxiety and depressive, internalizing/externalizing, and inattention symptoms, as predictors of treatment outcome in adolescents receiving intensive residential treatment for OCD. The sample comprised 314 adolescents aged 13-17 years with treatment-resistant OCD and a Children's Yale-Brown Obsessive-Compulsive Scale Self-Report (CY-BOCS-SR) total score ≥16. Bivariate and multiple regression models were used to evaluate the predictors of continuous OCD severity outcome and treatment response. Results of the bivariate regression analyses of predictors demonstrated that length of treatment, pre-treatment OCD severity, and symptoms of anxiety and depression significantly predicted post-treatment OCD severity, while only symptoms of depression and anxiety predicted treatment response. When including all predictors in the same model, only baseline OCD severity remained a significant predictor of post-treatment OCD severity, and none of the assessed variables significantly predicted treatment response. Results indicate that low pre-treatment OCD severity predicts lower OCD severity following treatment, although it did not predict treatment response.

Cost-Effectiveness of Treatment Alternatives for Treatment-Refractory Pediatric Obsessive-Compulsive Disorder.

PURPOSE: Current guidelines for first-line treatment of childhood OCD are cognitive-behavioral therapy (CBT) utilizing exposure and response prevention (ERP), and/or antidepressant (ADM) pharmacotherapy, specifically serotonin reuptake inhibitors (SRI). Given that first-line are relatively similar in terms of clinical effectiveness, the role of costs to provide such services may help influence treatment decisions. In the case of treatment refractory pediatric OCD, this cost-effectiveness analysis (CEA) aims to further evaluate two additional, higher intensity combination therapies, namely OCD-specific Intensive Outpatient (IOP) and Partial Hospitalization Programs (PHP), to determine the additional benefits, in terms of effectiveness, that may result, and the corresponding increase in costs for these higher-intensity courses of therapy. RESULTS: IOP was the most cost-effective strategy in terms of change in CY-BOCS, pre/post treatment, equal to 16.42 units, followed by PHP and CBT monotherapy augmented with ADM CBT-monotherapy augmented with additional CBT and ADM-only augmented with CBT followed closely with 15.56 and 14.75 unit improvements in CY-BOCS. IOP accomplished its superior cost-effectiveness with an Incremental Cost-Effectiveness Ratio (ICER), of $48,834, lower than either of the established willingness to Pay thresholds. CONCLUSIONS: Lack of access to high fidelity, high dose CBT paired with pharmacotherapy is an issue for OCD patients and families. Among youth who were treatment non-responsive, these results indicate the superiority of a high dosage CBT strategy, indicating the need to increase access to these treatments.

Obsessive-compulsive symptoms in eating disorders: A network investigation.

OBJECTIVE: Eating disorders (EDs) are complex, heterogeneous, and severe psychiatric syndromes. They are highly comorbid with obsessive-compulsive disorder (OCD) which exacerbates the course of illness and impedes treatment. However, the direct functional relations between EDs and OCD symptoms remain largely unexplored. Hence, using network analysis, we investigated the relationship between ED and OCD at the level of symptoms in a heterogeneous clinical sample. METHOD: We used cross sectional data of 303 treatment-seeking patients with clinically relevant ED and OCD pathology. We constructed a regularized partial correlation network that featured both ED and OCD symptoms as nodes. To determine each symptom's influence, we calculated expected influence (EI) as an index of symptom centrality (i.e., "importance"). Bridge symptoms (i.e., symptoms from one syndromic cluster that have strong connections to symptoms of another syndromic cluster) were identified by computing bridge expected influence metrics. RESULTS: Fear of weight gain and dietary restraint were especially important among the ED symptoms. Interference due to obsessions was the key feature of OCD. ED and OCD clustered distinctly with few potential bridges between clusters. DISCUSSION: This study underscores the importance of cognitive symptoms for both ED and OCD although direct functional links between the two clusters are missing. Potentially, a network incorporating nodes capturing features of personality may account for diagnostic comorbidity better than specific symptoms of EDs or features of OCD do.

Treatments used for obsessive-compulsive disorder-An international perspective.

OBJECTIVE: The objective of this study was to characterise international trends in the use of psychotropic medication, psychological therapies, and novel therapies used to treat obsessive-compulsive disorder (OCD). METHODS: Researchers in the field of OCD were invited to contribute summary statistics on the characteristics of their samples. Consistency of summary statistics across countries was evaluated. RESULTS: The study surveyed 19 expert centres from 15 countries (Argentina, Australia, Brazil, China, Germany, Greece, India, Italy, Japan, Mexico, Portugal, South Africa, Spain, the United Kingdom, and the United States) providing a total sample of 7,340 participants. Fluoxetine (n = 972; 13.2%) and fluvoxamine (n = 913; 12.4%) were the most commonly used selective serotonin reuptake inhibitor medications. Risperidone (n = 428; 7.3%) and aripiprazole (n = 415; 7.1%) were the most commonly used antipsychotic agents. Neurostimulation techniques such as transcranial magnetic stimulation, deep brain stimulation, gamma knife surgery, and psychosurgery were used in less than 1% of the sample. There was significant variation in the use and accessibility of exposure and response prevention for OCD. CONCLUSIONS: The variation between countries in treatments used for OCD needs further evaluation. Exposure and response prevention is not used as frequently as guidelines suggest and appears difficult to access in most countries. Updated treatment guidelines are recommended.

Single chain variable fragment antibodies directed against SOD1 ameliorate disease in mutant SOD1 transgenic mice.

Mutations in Cu/Zn superoxide dismutase (SOD1) are the cause of ~20% of cases of familial ALS (FALS), which comprise ~10% of the overall total number of cases of ALS. Mutant (mt) SOD1 is thought to cause FALS through a gain and not loss in function, perhaps as a result of the mutant protein's misfolding and aggregation. Previously we used a phage display library to raise single chain variable fragment antibodies (scFvs) against SOD1, which were found to decrease aggregation of mtSOD1 and toxicity in vitro. In the present study, we show that two scFvs directed against SOD1 ameliorate disease in G93A mtSOD1 transgenic mice and also decrease motor neuron loss, microgliosis, astrocytosis, as well as SOD1 burden and aggregation. The results suggest that the use of antibodies or antibody mimetics directed against SOD1 may be a useful therapeutic direction in mtSOD1-induced FALS. Since studies suggest that wild type SOD1 may be misfolded similar to that seen with mtSOD1, this therapeutic direction may be effective in sporadic as well as FALS.

A Recombinant Affinity Reagent Specific for a Phosphoepitope of Akt1.

The serine/threonine-protein kinase, Akt1, plays an important part in mammalian cell growth, proliferation, migration and angiogenesis, and becomes activated through phosphorylation. To monitor phosphorylation of threonine 308 in Akt1, we developed a recombinant phosphothreonine-binding domain (pTBD) that is highly selective for the Akt1 phosphopeptide. A phage-display library of variants of the Forkhead-associated 1 (FHA1) domain of yeast Rad53p was screened by affinity selection to the phosphopeptide, 301-KDGATMKpTFCGTPEY-315, and yielded 12 binding clones. The strongest binders have equilibrium dissociation constants of 160⁻180 nanomolar and are phosphothreonine-specific in binding. The specificity of one Akt1-pTBD was compared to commercially available polyclonal antibodies (pAbs) generated against the same phosphopeptide. The Akt1-pTBD was either equal to or better than three pAbs in detecting the Akt1 pT308 phosphopeptide in ELISAs.

Gender differences in eating disorder psychopathology across DSM-5 severity categories of anorexia nervosa and bulimia nervosa.

OBJECTIVE: This study examined whether patterns of eating-disorder (ED) psychopathology differed by gender across DSM-5 severity specifiers in anorexia nervosa (AN) and bulimia nervosa (BN). METHOD: We tested whether ED psychopathology differed across DSM-5 severity specifiers among 532 adults (76% female) in a residential treatment center with AN or BN. We hypothesized that severity of ED psychopathology would increase in tandem with increasing severity classifications for both males and females with AN and BN. RESULTS: Among females with BN, DSM-5 severity categories were significantly associated with increasing ED psychopathology, including Eating Disorder Examination-Questionnaire dietary restraint, eating concern, shape concern, and weight concern; and Eating Disorder Inventory drive for thinness and bulimia. ED psychopathology did not differ across DSM-5 severity levels for males with BN. For both males and females with AN, there were no differences in ED psychopathology across severity levels. DISCUSSION: Results demonstrate that DSM-5 severity specifiers may function differently for males versus females with BN. Taken together, data suggest DSM-5 severity specifiers may not adequately capture severity, as intended, for males with BN and all with AN. Future research should evaluate additional clinical validators of DSM-5 severity categories (e.g., chronicity, treatment non-response), and consider alternate classification schemes.

Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3.

Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated "MLK3 SH3-interacting peptide" (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a ß-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1-4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.