Robust Chemoenzymatic Synthesis of Keratinimicin Aglycone Analogues Facilitated by the Structure and Selectivity of OxyB.

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is 5-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether cross-link on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

Context-dependent requirement of G protein coupling for Latrophilin-2 in target selection of hippocampal axons.

The formation of neural circuits requires extensive interactions of cell-surface proteins to guide axons to their correct target neurons. Trans-cellular interactions of the adhesion G protein-coupled receptor latrophilin-2 (Lphn2) with its partner teneurin-3 instruct the precise assembly of hippocampal networks by reciprocal repulsion. Lphn2 acts as a repulsive receptor in distal CA1 neurons to direct their axons to the proximal subiculum, and as a repulsive ligand in the proximal subiculum to direct proximal CA1 axons to the distal subiculum. It remains unclear if Lphn2-mediated intracellular signaling is required for its role in either context. Here, we show that Lphn2 couples to Gα12/13 in heterologous cells; this coupling is increased by constitutive exposure of the tethered agonist. Specific mutations of Lphn2's tethered agonist region disrupt its G protein coupling and autoproteolytic cleavage, whereas mutating the autoproteolytic cleavage site alone prevents cleavage but preserves a functional tethered agonist. Using an in vivo misexpression assay, we demonstrate that wild-type Lphn2 misdirects proximal CA1 axons to the proximal subiculum and that Lphn2 tethered agonist activity is required for its role as a repulsive receptor in axons. By contrast, neither tethered agonist activity nor autoproteolysis were necessary for Lphn2's role as a repulsive ligand in the subiculum target neurons. Thus, tethered agonist activity is required for Lphn2-mediated neural circuit assembly in a context-dependent manner.

The complex brain circuits that allow animals to sense and interact with their environment start to form early during development. Throughout this period, neurons extend fiber-like projections to establish precise wiring patterns. Various types of proteins at the surface of both incoming fibers and target cells ensure that only the right partners will connect together. Latrophilin-2, for example, is a neuronal surface protein essential for the formation of accurate connections in the hippocampus, a brain region important for memory. Studded through the membrane of certain neurons, it acts as a signal-sending ligand to direct incoming fibers, with neurons that carry Latrophilin-2 repelling projections from cells that display certain protein partners. At the same time, Latrophilin-2 also allows neurons to receive chemical signals by working with intracellular signaling proteins known as G proteins, which help to relay information between cells. It remained unclear how this role as a signalling receptor participates in the wiring of the hippocampus during development. To explore this question, Pederick, Perry-Hauser et al. examined the impact of Latrophilin-2 on the connection patterns of mouse hippocampal neurons that do not normally carry this protein. Introducing Latrophilin-2 into these 'proximal CA1 cells' misdirected them away from their usual partners ­ unless Latrophilin-2 was altered so that it could not interact with G proteins. In contrast, forcing the connecting partners of CA1 cells to display normal or altered versions of Latrophilin-2 did not interfere with the protein acting as a repulsive ligand. Taken together, these results suggest that the ability of Latrophilin-2 to signal through G proteins is important for neurons that are attempting to project their fibers onto other cells, but not important when Latrophilin-2 acts in targets to direct incoming fibers from other neurons. These results show that a single protein can shape neural circuits by acting both as a signal-receiving receptor and a signal-sending ligand depending on the context. In the future, Pederick, Perry-Hauser et al. hope that their findings will shed new light on how the wiring of the brain is disrupted in neurodevelopmental disorders.

Disentangling autoproteolytic cleavage from tethered agonist-dependent activation of the adhesion receptor ADGRL3.

Adhesion G protein-coupled receptor latrophilin 3 (ADGRL3), a cell adhesion molecule highly expressed in the central nervous system, acts in synapse formation through trans interactions with its ligands. It is largely unknown if these interactions serve a purely adhesive function or can modulate G protein signaling. To assess how different structural elements of ADGRL3 (e.g., the adhesive domains, autoproteolytic cleavage site, or tethered agonist (TA)) impact receptor function, we require constructs that disrupt specific receptor features without impacting others. While we showed previously that mutating conserved Phe and Met residues in the TA of ADGRL3-C-terminal fragment (CTF), a CTF truncated to the G protein-coupled receptor proteolysis site, abolishes receptor-mediated G protein activation, we now find that autoproteolytic cleavage is disrupted in the full-length version of this construct. To identify a construct that disrupts TA-dependent activity without impacting proteolysis, we explored other mutations in the TA. We found that mutating the sixth and seventh residues of the TA, Leu and Met, to Ala impaired activity in a serum response element activity assay for both full-length and CTF constructs. We confirmed this activity loss results from impaired G protein coupling using an assay that acutely exposes the TA through controlled proteolysis. The ADGRL3 mutant expresses normally at the cell surface, and immunoblotting shows that it undergoes normal autoproteolysis. Thus, we found a construct that disrupts tethered agonism while retaining autoproteolytic cleavage, providing a tool to disentangle these functions in vivo. Our approach and specific findings are likely to be broadly applicable to other adhesion receptors.

Biosynthesis of selenium-containing small molecules in diverse microorganisms.

Selenium is an essential micronutrient in diverse organisms. Two routes are known for its insertion into proteins and nucleic acids, via selenocysteine and 2-selenouridine, respectively1. However, despite its importance, pathways for specific incorporation of selenium into small molecules have remained elusive. Here we use a genome-mining strategy in various microorganisms to uncover a widespread three-gene cluster that encodes a dedicated pathway for producing selenoneine, the selenium analogue of the multifunctional molecule ergothioneine2,3. We elucidate the reactions of all three proteins and uncover two novel selenium-carbon bond-forming enzymes and the biosynthetic pathway for production of a selenosugar, which is an unexpected intermediate en route to the final product. Our findings expand the scope of biological selenium utilization, suggest that the selenometabolome is more diverse than previously thought, and set the stage for the discovery of other selenium-containing natural products.

Short Arrestin-3-Derived Peptides Activate JNK3 in Cells.

Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.

Evaluating an innovative integrated training for child and youth services: Lessons learned.

This article highlights one child and youth services agency's journey to develop and pilot a system for tracking training effectiveness for staff. Given the importance of intentional and strong staff training to the success of an agency, the purpose of this study was to report training outcomes across four-time intervals (i.e., immediately after training considered here as baseline, 6 months, 12 months, and 18 months) using a tracking system based on best practices in staff development. Results indicated that the tracking system allowed this agency to measure and describe post-training outcomes for staff across the four-time intervals. It can be inferred that such training positively influenced youth outcomes. Specifically, the training and follow-up expanded staff's ability to effectively internalize concepts and be more intentional with their time for the benefit of youth care.

The Two Non-Visual Arrestins Engage ERK2 Differently.

Arrestin binding to active phosphorylated G protein-coupled receptors terminates G protein coupling and initiates another wave of signaling. Among the effectors that bind directly to receptor-associated arrestins are extracellular signal-regulated kinases 1/2 (ERK1/2), which promote cellular proliferation and survival. Arrestins may also engage ERK1/2 in isolation in a pre- or post-signaling complex that is likely in equilibrium with the full signal initiation complex. Molecular details of these binary complexes remain unknown. Here, we investigate the molecular mechanisms whereby arrestin-2 and arrestin-3 (a.k.a. ß-arrestin1 and ß-arrestin2, respectively) engage ERK1/2 in pairwise interactions. We find that purified arrestin-3 binds ERK2 more avidly than arrestin-2. A combination of biophysical techniques and peptide array analysis demonstrates that the molecular basis in this difference of binding strength is that the two non-visual arrestins bind ERK2 via different parts of the molecule. We propose a structural model of the ERK2-arrestin-3 complex in solution using size-exclusion chromatography coupled to small angle X-ray scattering (SEC-SAXS). This binary complex exhibits conformational heterogeneity. We speculate that this drives the equilibrium either toward the full signaling complex with receptor-bound arrestin at the membrane or toward full dissociation in the cytoplasm. As ERK1/2 regulates cell migration, proliferation, and survival, understanding complexes that relate to its activation could be exploited to control cell fate.

Total Synthesis of Shearinines†D and G: A Convergent Approach to Indole Diterpenoids.

The first total syntheses of the indole diterpenoids (+)-shearinine G and D are disclosed. The successful routes rely on late-stage coupling of two complex fragments. Formation of the challenging trans-hydrindane motif was accomplished by diastereoselective, intramolecular cyclopropanation. A one-pot sequence consisting of Sharpless dihydroxylation/Achmatowicz reaction was developed to install the dioxabicyclo[3.2.1]octane motif. The indenone subunit was accessed by Prins cyclization. Tuning the electronic nature of the substituents on the parent arylcarboxaldehyde allowed access to divergent products that were further transformed into shearinines G and D. Riley-type oxidation of a bicyclic enone yielded a surprising stereochemical outcome.

Assays for detecting arrestin interaction with GPCRs.

The four vertebrate arrestins play a key role in the desensitization and internalization of G protein-coupled receptors (GPCRs) and also mediate receptor-dependent signaling. Recent work has shown that bias for arrestin vs G protein signaling could offer certain therapeutic advantages (or disadvantages) in different systems, making assays that measure arrestin binding to receptors important for drug discovery efforts. Herein, we briefly review several commonly used techniques for measuring arrestin binding to receptors, as well as provide an in-depth and methodologically focused review of two methods that do not require receptor modification. The first approach measures direct binding between purified arrestin and rhodopsin, and the second measures the recruitment of arrestin to receptors in living cells.

Rational, emotional, or both? Subcomponents of psychopathy predict opposing moral decisions.

Recent research has documented a small but significant correlation between psychopathic capacities and utilitarian moral judgment, although the findings are generally inconsistent and unclear. We propose that one way to make sense of mixed findings is to consider variation in perspective-taking capacities of psychopathic individuals. With this in mind, we had criminal offenders (n = 60), who varied in their psychopathy levels according to the Psychopathy Checklist-Revised (PCL-R), respond to common sacrificial moral dilemmas (e.g., trolley dilemmas) under different conditions. In a baseline condition, participants simply responded to the sacrificial moral dilemmas as is typically done in previous research. In an "emotion-salient" condition, participants had to reason about the emotions of another person after solving moral dilemmas (deliberative processing). In the "emotion-ambiguous" condition, participants saw images of people in distress, after solving moral dilemmas, but did not have to explicitly reason about such emotions (spontaneous processing). The four PCL-R facets predicted distinct interference effects depending on spontaneous versus deliberative processing of hypothetical victim's emotions. The findings suggest that the use of a multi-faceted approach to account for cognitive and moral correlates of psychopathy may help address previously mixed results. Implications and future directions for theory and research are discussed.

Viewpoints on the First Transatlantic GPCR Symposium for Early-Career Investigators.

In July 2021, we organized a virtual symposium aimed at early-career investigators (ECIs) in G protein-coupled receptor (GPCR) research: the first Transatlantic ECI GPCR Symposium. Here, we discuss the proceedings of this symposium and the unique networking events with GPCR leaders including the Nobel Laureates Dr. Robert Lefkowitz and Dr. Brian Kobilka.

Detection of Psychopathic Traits in Emotional Faces.

When meeting someone at zero acquaintance, we make assumptions about each other that encompass emotional states, personality traits, and even cognitive abilities. Evidence suggests individuals can accurately detect psychopathic personality traits in strangers based on short video clips or photographs of faces. We present an in-depth examination of this ability. In two studies, we investigated whether high psychopathy traits are perceivable and whether other traits affect ratings of psychopathic traits in the sense of a halo effect. On the perceiver's end, we additionally examined how cognitive abilities and personality traits of the responders affect these ratings. In two studies (n1 = 170 community adults from the USA, n2 = 126 students from Australia), participants rated several targets on several characteristics of psychopathy, as well as on attractiveness, masculinity, sympathy, trustworthiness, neuroticism, intelligence, and extraversion. Results show that responders were generally able to detect psychopathy. Responders generally came to a consensus in their ratings, and using profile similarity metrics, we found a weak relation between ratings of psychopathy and the targets' psychopathy level as determined by the Psychopathy Checklist: Short Version. Trait ratings, though, were influenced by the ratings of other traits like attractiveness. Finally, we found accuracy in the perception of psychopathy was positively related to fluid intelligence but unrelated to emotion perception ability.

Emotion expression abilities and psychopathy.

The ability to express emotion is considered a core socioemotional skill; however, most research is focused on receptive abilities, with little investigation of productive abilities. We present an investigation of individual differences in facial expression of emotion using observational techniques. Given descriptions of highly psychopathic persons as successful liars and manipulators, we investigate the ability to intentionally pose emotional expressions when no emotion is elicited. A mixed sample of adult men (N = 316 community volunteers, prison inmates, and forensic-psychiatric patients) ranging along the psychopathy continuum were asked to facially express a nonfelt emotion, specifically anger, disgust, fear, happiness, sadness, and surprise, through either written instructions or through imitation of a target's facial expression. Through structural equation modeling, we evaluate relations between this emotion expression ability and general mental ability, interpersonal abilities, and psychopathy. We find that psychopathy is moderately associated with poorer emotion expression ability, meaning highly psychopathic individuals are poorer at imitating the expressions of others and poorer at expressing all emotions. However, this deficit is largely attributable to deficits in general mental ability. These results challenge the view that highly psychopathic individuals have the cognitive skills to support a superior ability to deceive or manipulate others. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

The Serendipitous Discovery of a Rose Odorant.

Serendipity has played a role in many groundbreaking scientific discoveries. Key to their identification and exploitation is the ability to recognize the unexpected and invest time trying to understand it. Like any other field of scientific research, total synthesis requires determination and perseverance. When the first-generation route towards a target compound fails, new approaches are developed based on insights gained in the initial studies. Careful analysis of data obtained in a 'failed' approach, e.g. when a reaction did not yield the desired or any expected outcome, can lead to spectacularly improved routes and discoveries that have impact beyond the synthesis of the selected target compound. Serendipity has further led to the identification of intriguing properties that materials or single molecules have, as exemplified by the discovery of electrically conductive polymers. During our total synthesis endeavors towards a complex natural product, we identified a small molecule with interesting olfactory properties, which we decided to investigate further.

A logic model development for an adolescent based intervention to improve benefits from Therapeutic Residential Care (TRC).

There is a significant need to comprehensively describe and illustrate via a logic model what processes work for adolescents in residential treatment facilities and how to make improvements (Bean, White, Neagle & Lake, 2005).The purpose of this article is to highlight one Adolescent TRC's journey to develop and implement a working Logic Model.

Nursing genetics and genomics: The International Society of Nurses in Genetics (ISONG) survey.

BACKGROUND: The International Society of Nursing in Genetics (ISONG) fosters scientific and professional development in the discovery, interpretation, and application of genomic information in nursing research, education, and clinical practice. OBJECTIVES: Assess genomic-related activities of ISONG members in research, education and practice, and competencies to serve as global leaders in genomics. DESIGN: Cross-sectional survey (21-items) assessing genomic-related training, knowledge, and practice. SETTINGS: An email invitation included a link to the anonymous online survey. PARTICIPANTS: All ISONG members (n = 350 globally) were invited to partake. METHODS: Descriptive statistics and Wilcoxon Rank Sum Test for between-group comparisons. RESULTS: Respondents (n = 231, 66%), were mostly Caucasian, female, with a master's degree or higher. Approximately 70% wanted to incorporate genomics in research, teaching, and practice. More than half reported high genomic competency, and over 95% reported that genomics is relevant the next 5 years. CONCLUSIONS: Findings provide a foundation for developing additional educational programs for an international nursing workforce in genomics.

Evaluating the Utility of mHealth ECG Heart Monitoring for the Detection and Management of Atrial Fibrillation in Clinical Practice.

BACKGROUND: Little attention has focused on the integration of mobile health (mHealth) technology with self-management approaches to improve the detection and management of atrial fibrillation (AF) in clinical practice. OBJECTIVE: The objective of this study was to investigate the differences between mHealth and usual care over a 6-month follow-up period among patients with a known history of atrial fibrillation. METHODS: A pilot cohort from within the larger ongoing randomized trial, iPhone® Helping Evaluate Atrial fibrillation Rhythm through Technology (iHEART), was evaluated to determine differences in detection of AF and atrial flutter (AFL) recurrence rates (following treatment to restore normal rhythm) between patients undergoing daily smartphone ECG monitoring and age and gender matched control patients. SF-36v2TM QoL assessments were administered at baseline and 6 months to a subset of the patients undergoing daily ECG monitoring. Differences between groups were assessed by t-test, Fisher's exact test, and Cox proportional hazard models. RESULTS: Among the 23 patients with smartphone ECG monitors (16 males and 7 females, mean age 55 ± 10), 14 (61%) had detection of recurrent AF/AFL versus 30% of controls. During the follow-up period, patients given smartphone ECG monitors were more than twice as likely to have an episode of recurrent AF/AFL detected (hazard ratio: 2.55; 95% CI: 1.06 - 6.11; p = 0.04). Among the 13 patients with baseline and 6 month QoL assessments, significant improvements were observed in the physical functioning (p = 0.009), role physical (p = 0.007), vitality (p = 0.03), and mental health domains (p = 0.02). CONCLUSIONS: Cardiac mHealth self-monitoring is a feasible and effective mechanism for enhancing AF/AFL detection that improves quality of life.

Clinical and gender differences in heart transplant recipients in the NEW HEART study.

BACKGROUND: Little attention has focused on gender differences in cardiac comorbidities and outcomes in patients undergoing orthotropic heart transplant. OBJECTIVE: The objective of this study was to investigate gender differences at baseline and during follow-up among heart transplant patients. METHODS: An observational cohort within the NEW HEART study was evaluated to determine gender differences in relation to age, coexisting cardiac comorbidities, and outcomes. Differences were assessed by t-test, Fisher's exact test, and logistic regression analysis. RESULTS: Male transplant recipients ( n = 238) were significantly older than female recipients ( n = 92), with a greater percentage over 60 years of age (45% vs. 24%, p = 0.0006). Males were more likely to have hypertension (63% vs. 49%, p = 0.034), dyslipidemia (62% vs. 45%, p = 0.006), a history of smoking (52% vs. 35%, p = 0.009), and diabetes (42% vs. 21%, p = 0.0002). Analysis of endomyocardial biopsies obtained during the 1-year follow-up period demonstrated that women averaged more episodes of acute rejection than men (3.9 vs. 3.0, p = 0.009). While most episodes of rejection were mild, women were more likely than men to have episodes of moderate or severe rejection (14% vs. 5%, p = 0.012) and to be hospitalized for acute rejection (15% vs. 6%, p = 0.013). There were no significant differences in mortality. CONCLUSIONS: Men were more likely than women to be older and to have diabetes, dyslipidemia, hypertension, and a history of smoking. Women were more likely to experience moderate or severe allograft rejection and to be hospitalized for acute rejection. Future investigation of the reasons for these gender differences is warranted and may improve clinical care of women undergoing cardiac transplantation.

A single-center randomized, controlled trial investigating the efficacy of a mHealth ECG technology intervention to improve the detection of atrial fibrillation: the iHEART study protocol.

BACKGROUND: Atrial fibrillation is a major public health problem and is the most common cardiac arrhythmia, affecting an estimated 2.7 million Americans. The true prevalence of atrial fibrillation is likely underestimated because episodes are often sporadic; therefore, it is challenging to detect and record an occurrence in a "real world" setting. To date, mobile health tools that promote earlier detection and treatment of atrial fibrillation and improvement in self-management behaviors and knowledge have not been evaluated. This study will be the first to address the epidemic problem of atrial fibrillation with a novel approach utilizing advancements in mobile health electrocardiogram technology to empower patients to actively engage in their healthcare and to evaluate impact on quality of life and quality-adjusted life years. Furthermore, sending a daily electrocardiogram transmission, coupled with receiving educational and motivational text messages aimed at promoting self-management and a healthy lifestyle may improve the management of chronic cardiovascular conditions (e.g., hypertension, diabetes, heart failure, etc.). Therefore, we are currently conducting a randomized controlled trial to assess the efficacy of a mobile health intervention, iPhone® Helping Evaluate Atrial fibrillation Rhythm through Technology (iHEART) versus usual cardiac care. METHODS: The iHEART study is a single center, prospective, randomized controlled trial. A total of 300 participants with a recent history of atrial fibrillation will be enrolled. Participants will be randomized 1:1 to receive the iHEART intervention, receiving an iPhone® equipped with an AliveCor® Mobile ECG and accompanying Kardia application and behavioral altering motivational text messages or usual cardiac care for 6 months. DISCUSSION: This will be the first study to investigate the utility of a mobile health intervention in a "real world" setting. We will evaluate the ability of the iHEART intervention to improve the detection and treatment of recurrent atrial fibrillation and assess the intervention's impact on improving clinical outcomes, quality of life, quality-adjusted life-years and disease-specific knowledge. TRIAL REGISTRATION: NCT02731326 ; Verified April 2016.