Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a potential redox-regulated function.

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.

When social hierarchy matters grammatically: Investigation of the processing of honorifics in Korean.

Korean grammar encodes relative social hierarchies among interlocutors in various ways. This study utilized honorific subject-verb agreement in Korean to investigate how social hierarchies are processed during sentence comprehension. The experimental results showed that honorific violations elicited processing difficulties. The use of an honorific verb with an unhonorifiable subject resulted in lower naturalness ratings, longer reading times, and elicited a P600, similar to effects observed with number, person, and gender agreement in Spanish or English. These findings suggest that social hierarchies have become integrated into grammar, constraining how native Korean speakers process sentences. However, the agreement between honorific subjects and verbs seems asymmetrical; the mismatch effect was smaller or absent when an honorifiable subject was not accompanied by an honorific verb, suggesting that while an honorific verb requires an honorifiable subject, the reverse is not necessarily true. The results indicate that the -si agreement in Korean is a form of morpho-syntactic agreement, despite its asymmetrical nature.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors.

The specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEP) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEP promotes Mk specification, while pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkP) and E progenitors (ErP), there are higher levels of serine-phosphorylated RUNX1 in MkP than ErP, and mutant RUNX1 with phospho-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we employ human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and non-phosphorylatable (RUNX1-4A) mutants showing that the three forms of RUNX1 differentially regulate expression of 2,625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D-expressing HEL cells and those upregulated in primary human MkP versus MEP. While inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEP, specific inhibition of CDK9 in MEP leads to both decreased RUNX1 phosphorylation and increased erythroid commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell-type specific transcription factor.

Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

Where do psychologists turn to inform clinical decisions? Audience segmentation to guide dissemination strategies.

Background: Audience segmentation is an analysis technique that can identify meaningful subgroups within a population to inform the tailoring of dissemination strategies. We have conducted an empirical clustering audience segmentation study of licensed psychologists using survey data about the sources of knowledge they report most often consulting to guide their clinical decision-making. We identify meaningful subgroups within the population and inform the tailoring of dissemination strategies for evidence-based practice (EBP) materials. Method: Data come from a 2018-2019 web-based survey of licensed psychologists who were members of the American Psychological Association (APA; N = 518, response rate = 29.8%). Ten dichotomous variables assessed sources that psychologists regularly consult to inform clinical decision-making (e.g., colleagues, academic literature, and practice guidelines). We used latent class analysis to identify segments of psychologists who turn to similar sources and named each segment based on the segment's most salient characteristics. Results: Four audience segments were identified: the No-guidelines (45% of psychologists), Research-driven (16%), Thirsty-for-knowledge (9%), and No-reviews (30%). The four segments differed not only in their preferred sources of knowledge, but also in the types of evidence-based posttraumatic stress disorder (PTSD) treatments they provide, their awareness and usage intention of the APA PTSD clinical practice guideline, and attitudes toward clinical practice guidelines. Conclusion: The results demonstrate that licensed psychologists are heterogeneous in terms of their knowledge-seeking behaviors and preferences for knowledge sources. The distinctive characteristics of these segments could guide the tailoring of dissemination materials and strategies to subsequently enhance the implementation of EBP among psychologists.

Audience segmentation is a dissemination strategy that categorizes a group of intended users or audience into meaningful subgroups based on their beliefs, behaviors, and/or other characteristics. Like many other scientific or medical fields, clinical psychology also struggles to use clinically tested psychological treatments (or EBPs) in everyday practice due to practical challenges. To help address such barriers, professional organizations like the American Psychological Association (APA) publish clinical practice guidelines that practitioners can use to learn more about EBPs. However, even these clinical practice guidelines are not often used, so this study employed the audience segmentation analysis to better understand psychologists' diverse attitudes, behaviors, and preferences regarding clinical practice guidelines and other clinical information sources. Our study found four distinct subgroups within approximately 600 APA-registered psychologists based on their preferred source of knowledge: the no-guidelines (45% of psychologists), research-driven (16%), thirsty-for-knowledge (9%), and no-reviews (30%). Each subgroup also varied in the types of evidence-based treatments they provide, as well as their awareness, willingness to use, and attitudes toward clinical practice guidelines. This result shows that licensed psychologists are not a uniform group and that dissemination strategies should be adjusted to each subgroup's characteristics to maximize the effort to increase the use of EBPs among psychologists.

Building an Integrated Data Infrastructure to Examine the Spectrum of Suicide Risk Factors in Philadelphia Medicaid.

While there are many data-driven approaches to identifying individuals at risk of suicide, they tend to focus on clinical risk factors, such as previous psychiatric hospitalizations, and rarely include risk factors that occur in nonclinical settings, such as jails or emergency shelters. A better understanding of system-level encounters by individuals at risk of suicide could help inform suicide prevention efforts. In Philadelphia, we built a community-level data infrastructure that encompassed suicide death records, behavioral health claims, incarceration episodes, emergency housing episodes, and involuntary commitment petitions to examine a broader spectrum of suicide risk factors. Here, we describe the development of the data infrastructure, present key trends in suicide deaths in Philadelphia, and, for the Medicaid-eligible population, determine whether suicide decedents were more likely to interact with the behavioral health, carceral, and housing service systems compared to Medicaid-eligible Philadelphians who did not die by suicide. Between 2003 and 2018, there was an increase in the number of annual suicide deaths among Medicaid-eligible individuals, in part due to changes in Medicaid eligibility. There were disproportionately more suicide deaths among Black and Hispanic individuals who were Medicaid-eligible, who were younger on average, compared to suicide decedents who were never Medicaid-eligible. However, when we accounted for the racial and ethnic composition of the Medicaid population at large, we found that White individuals were four times as likely to die by suicide, while Asian, Black, Hispanic, and individuals of other races were less likely to die by suicide. Overall, 58% of individuals who were Medicaid-eligible and died by suicide had at least one Medicaid-funded behavioral health claim, 10% had at least one emergency housing episode, 25% had at least one incarceration episode, and 22% had at least one involuntary commitment. By developing a data infrastructure that can incorporate a broader spectrum of risk factors for suicide, we demonstrate how communities can harness administrative data to inform suicide prevention efforts. Our findings point to the need for suicide prevention in nonclinical settings such as jails and emergency shelters, and demonstrate important trends in suicide deaths in the Medicaid population.

Implementing a resilience coach program to support first year housestaff during the COVID-19 pandemic: early pilot results and comparison with non-housestaff sessions.

BACKGROUND: In response to the COVID-19 pandemic, we launched the Penn Medicine Coping First Aid program to provide psychosocial supports to our health system community. Our approach leveraged lay health worker volunteers trained in principles of Psychological First Aid to deliver coaching services through a centralized virtual platform. METHODS: We emailed all (n = 408) first year housestaff (i.e., residents and fellows) with an invitation to schedule a session with a resilience coach. We compared the mental health concerns, symptoms, and Psychological First Aid techniques recorded in (n = 67) first year housestaff sessions with (n = 91) sessions of other employees in the health system. RESULTS: Between June and November 2020, forty-six first year housestaff attended at least one resilience coaching session. First year housestaff most commonly presented with feelings of anxiety and sadness and shared concerns related to the availability of social support. Resilience coaches most frequently provided practical assistance and ensured safety and comfort to first year housestaff. First year housestaff reported fewer physical or mental health symptoms and held shorter sessions with resilience coaches than non-housestaff. CONCLUSIONS: This work offers insights on how to address psychosocial functioning through low-intensity interventions delivered by lay personnel. More research is needed to understand the efficacy of this program and how best to engage housestaff in wellness and resilience programs throughout training, both during and beyond COVID-19.

Not in my treatment center: Leadership's perception of barriers to MOUD adoption.

INTRODUCTION: Despite their well-established effectiveness, medications for opioid use disorder (MOUD) are widely underutilized across the United States. In the context of a large publicly funded behavioral health system, we examined the relationship between a range of implementation barriers and a substance use disorder treatment agency's level of adoption of MOUD. METHODS: We surveyed leadership of publicly funded substance use disorder treatment centers in Philadelphia about the significance of barriers to implementing MOUD related to their workforce, organization, funding, regulations, and beliefs about MOUD's efficacy and safety. We queried leaders on the percentage of their patients with opioid use disorder who receive MOUD and examined associations between implementation barriers and MOUD adoption. RESULTS: Ratings of regulatory, organizational, or funding barriers of respondents who led high MOUD adopting agencies (N = 20) were indistinguishable from those who led agencies that were low adopting of MOUD (N = 23). In contrast, agency leaders who denied MOUD-belief or workforce barriers were significantly more likely to lead high-MOUD-adopting organizations. CONCLUSIONS: These findings suggest that leadership beliefs about MOUD may be a key factor of the organizational decision to adopt and should be a target of implementation efforts to increase direct provision of these medications.

Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.