Probing Bioactive Chemical Space to Discover RNA-Targeted Small Molecules.

Small molecules have become increasingly recognized as invaluable tools to study RNA structure and function and to develop RNA-targeted therapeutics. To rationally design RNA-targeting ligands, a comprehensive understanding and explicit testing of small molecule properties that govern molecular recognition is crucial. To date, most studies have primarily evaluated properties of small molecules that bind RNA in vitro, with little to no assessment of properties that are distinct to selective and bioactive RNA-targeted ligands. Therefore, we curated an RNA-focused library, termed the Duke RNA-Targeted Library (DRTL), that was biased towards the physicochemical and structural properties of biologically active and non-ribosomal RNA-targeted small molecules. The DRTL represents one of the largest academic RNA-focused small molecule libraries curated to date with more than 800 small molecules. These ligands were selected using computational approaches that measure similarity to known bioactive RNA ligands and that diversify the molecules within this space. We evaluated DRTL binding in vitro to a panel of four RNAs using two optimized fluorescent indicator displacement assays, and we successfully identified multiple small molecule hits, including several novel scaffolds for RNA. The DRTL has and will continue to provide insights into biologically relevant RNA chemical space, such as the identification of additional RNA-privileged scaffolds and validation of RNA-privileged molecular features. Future DRTL screening will focus on expanding both the targets and assays used, and we welcome collaboration from the scientific community. We envision that the DRTL will be a valuable resource for the discovery of RNA-targeted chemical probes and therapeutic leads.

Effects of an Adapted Telehealth Training Curriculum for Registered Behavior Technicians®.

Objectives: We evaluated a telehealth training curriculum to prepare Registered Behavior Technicians® (RBTs®) to conduct caregiver coaching on behavior analytic skills via telehealth. Methods: A non-concurrent multiple baseline across therapists design was used to evaluate the training procedures delivered by Board Certified Behavior Analysts® (BCBAs®). All therapists (RBTs) worked with one family during the baseline and training phases and an additional session was conducted with a different family during the novelty probe condition to see if therapists were able to perform the newly acquired skills without BCBA support. Results: Seven therapists who received behavioral skills training on the curriculum reached mastery criteria within three sessions. All RBTs were able to provide caregiver coaching via telehealth with high degrees of fidelity and were able to independently conduct a telehealth session with a new family without support from the BCBA. Conclusions: Results of the current study provide support for the use of the telehealth training curriculum to prepare RBTs to conduct ABA services via telehealth.

A Qualitative Review of Barriers and Facilitators Identified While Implementing the Native Students Together Against Negative Decisions Curriculum in a Multisite Dissemination and Implementation Study.

Culturally-adapted evidence-based programs (EBPs) are needed to promote healthy behaviors among Native teens and young adults. Little is known about the facilitators and barriers of implementing and sustaining EBPs in Native communities. This paper aims to identify those factors described by educators who implemented the Native Students Together Against Negative Decisions (STAND) curriculum. METHODS: We conducted qualitative, semi-structured interviews with 44 Native STAND educators from 48 sites throughout the United States. We used a modified grounded theory approach to explore barriers, facilitators, and sustainability factors related to implementing Native STAND. RESULTS: We learned that disruptions to staffing, coordination, and organizational factors were the most common barriers. Factors that improved implementation success included: tailoring the program to local needs/constraints, having a supportive Project Manager, improved fidelity due to check-in calls, and participation in summer training. Factors that improved sustainability included: access to needed infrastructure, administrative support, community support, and student interest. DISCUSSION: The delivery of Native STAND was further improved by person-to-person communication and resource sharing across sites. Sustaining EBPs in AI/AN settings requires culturally-tailored technical assistance, sufficient implementation funds for materials and staffing, and a community of peer educators to inspire forward progress. CONCLUSION: EBPs that reflect the needs and experiences of American Indian and Alaska Native (AI/AN) youth are necessary to address systemic inequities in adolescent health outcomes. The Native STAND Dissemination and Implementation study is among the first to assess facilitators and barriers to program delivery in diverse AI/AN settings.

Adding a One Health approach to a research framework for minority health and health disparities.

The National Institute on Minority Health and Health Disparities (NIMHD) has developed a framework to guide and orient research into health disparities and minority health. The framework depicts different domains of influence (such as biological and behavioral) and different levels of influence (such as individual and interpersonal). Here, influenced by the "One Health" approach, we propose adding two new levels of influence - interspecies and planetary - to this framework to reflect the interconnected nature of human, animal, and environmental health. Extending the framework in this way will help researchers to create new avenues of inquiry and encourage multidisciplinary collaborations. We then use the One Health approach to discuss how the COVID-19 pandemic has exacerbated health disparities, and show how the expanded framework can be applied to research into health disparities related to antimicrobial resistance and obesity.

Universal HIV screening in the emergency department: an interrupted time series analysis.

We performed a calendar-matched, 12-month, before (November 27, 2017 to November 26, 2018) and after (November 27, 2018 to November 26, 2019) study, to assess the utility of an emergency department-based HIV screening program. There were 710 and 14 335 patients screened for HIV during the pre and post-best practice alert (BPA) periods, respectively, representing more than a 20-fold increase in HIV screening following BPA implementation. Total HIV positive tests increased 5-fold following BPA implementation.

Psychometric evaluation of protective measures in Native STAND: A multi-site cross-sectional study of American Indian Alaska Native high school students.

American Indian and Alaska Native (AI/AN) youth are strong in culture and rich in heritage and experience unique strengths and challenges throughout adolescence. Documenting conditions that protect against risk factors associated with poor health outcomes are needed. We explored scales that measure self-esteem, culture, social support, and community from a sample of 1,456 youth involved in Native STAND, a culturally-relevant evidence-based sexual health intervention. We established content validity by reviewing existing literature and community feedback. Construct validity was examined using factor analysis. The final self-esteem model included seven items, factor loadings ranged from 0.47 to 0.63 for positive self-esteem and 0.77 to 0.81 for negative self-esteem. The final culture model included three items, factor loadings 0.73 to 0.89. The social support scale included four items, factor loadings ranged from 0.86 to 0.87 for family social support and 0.75 to 0.77 for friends social support. The community and community safety scale included three items; factor loadings ranged from 0.52 to 0.82. Coefficient alphas for scales ranged from α = 0.63 to α = 0.86. This study validated scales in a national sample of AI/AN youth-psychometric scales provide an essential tool for documenting the needs and strengths of AI/AN youth.

Identifying Associations in Minimum Inhibitory Concentration Values of Escherichia coli Samples Obtained From Weaned Dairy Heifers in California Using Bayesian Network Analysis.

Objective: Many antimicrobial resistance (AMR) studies in both human and veterinary medicine use traditional statistical methods that consider one bacteria and one antibiotic match at a time. A more robust analysis of AMR patterns in groups of animals is needed to improve on traditional methods examining antibiotic resistance profiles, the associations between the patterns of resistance or reduced susceptibility for all isolates in an investigation. The use of Bayesian network analysis can identify associations between distributions; this investigation seeks to add to the growing body of AMR pattern research by using Bayesian networks to identify relationships between susceptibility patterns in Escherichia coli (E. coli) isolates obtained from weaned dairy heifers in California. Methods: A retrospective data analysis was performed using data from rectal swab samples collected from 341 weaned dairy heifers on six farms in California and selectively cultured for E. coli. Antibiotic susceptibility tests for 281 isolates against 15 antibiotics were included. Bayesian networks were used to identify joint patterns of reduced susceptibility, defined as an increasing trend in the minimum inhibitory concentration (MIC) values. The analysis involved learning the network structure, identifying the best fitting graphical mode, and learning the parameters in the final model to quantify joint probabilities. Results: The graph identified that as susceptibility to one antibiotic decreases, so does susceptibility to other antibiotics in the same or similar class. The following antibiotics were connected in the final graphical model: ampicillin was connected to ceftiofur; spectinomycin was connected with trimethoprim-sulfamethoxazole, and this association was mediated by farm; florfenicol was connected with tetracycline. Conclusions: Bayesian network analysis can elucidate complex relationships between MIC patterns. MIC values may be associated within and between drug classes, and some associations may be correlated with farm of sample origin. Treating MICs as discretized variables and testing for joint associations in trends may overcome common research problems surrounding the lack of clinical breakpoints.

Are American Indian/Alaska Native Adolescent Health Behaviors Different? A Review of AI/AN Youth Involved in Native STAND Curriculum, 2014-2017 United States.

OBJECTIVES: To explore health behavior profiles of AI/AN youth involved in native students together against negative decisions (STAND), a national culture-based curriculum. METHODS: We analyzed data from 1236 surveys conducted among AI/AN youth at 40 native STAND implementation sites located in 16 states throughout the US from 2014 to 2017. Health profiles included demographics, sexual orientation, sexual activity, STI testing, cigarette use, and suicide attempts in the past 12-months. We used t-tests and chi square tests of independence to compare risk behavior prevalence among the sample. RESULTS: Health behavior profiles of AI/AN youth indicate that 45.6% of youth did not use condoms the last time they had sex, and 82.7% have never been tested for STIs. Differences in cigarette smoking were observed in questioning youth (questioning: 80.3%, straight/heterosexual: 63.8%, LGBTQ2S + : 49.9%, p = 0.03). CONCLUSIONS FOR PRACTICE: Health behaviors related to sex, substance, violence and self-harm, are at least as common for AI/AN youth as those observed in other US teens. Future research should consider similarities and differences in health profiles of AI/AN youth when designing interventions that affect them. Further, our findings underscore the need for culturally-relevant curricula like native STAND, not because their health behavior is different, but because their socio-ecologic environment is different.

The Role of Substrate Mediated Allostery in the Catalytic Competency of the Bacterial Oligosaccharyltransferase PglB.

The oligosaccharyltransferase of Campylobacter lari (PglB) catalyzes the glycosylation of asparagine in the consensus sequence N-X-S/T, where X is any residue except proline. Molecular dynamics simulations of PglB bound to two different substrates were used to characterize the differences in the structure and dynamics of the substrate-enzyme complexes that can explain the higher catalytic efficiency observed for substrates containing threonine at the +2 position rather than serine. We observed that a threonine-containing substrate is more tightly bound than a serine-containing substrate. Because serine lacks a methyl group relative to threonine, the serine-containing peptide cannot stably form simultaneous van der Waals interactions with T316 and I572 as the threonine-containing substrate can. As a result, the peptide-PglB interaction is destabilized and the allosteric communication between the periplasmic domain and external loop EL5 is disrupted. These changes ultimately lead to the reorientation of the periplasmic domain relative to the transmembrane domain such that the two domains are further apart compared to PglB bound to the threonine-containing peptide. The crystal structure of PglB bound to the peptide and a lipid-linked oligosaccharide analog shows a pronounced closing of the periplasmic domain over the transmembrane domain in comparison to structures of PglB with peptide only, indicating that a closed conformation of the domains is needed for catalysis. The results of our studies suggest that lower enzymatic activity observed for serine versus threonine results from a combination of less stable binding and structural changes in PglB that influence the ability to form a catalytically competent state. This study illustrates a mechanism for substrate specificity via modulation of dynamic allosteric pathways.

ED syphilis and gonorrhea/chlamydia cotesting practices before and after the implementation of an electronic health record-based alert.

BACKGROUND: The prevalence of syphilis is increasing in many countries, including the USA. The ED is often used by underserved populations, making it an important setting to test and treat patients who are not evaluated in outpatient clinical settings. We aimed to assess the utility of an ED-based syphilis and gonorrhoea/chlamydia cotesting protocol by comparing testing practices before and after its implementation. METHODS: We implemented an electronic health record (EHR) alert that prompted clinicians to order syphilis testing in patients undergoing gonorrhoea/chlamydia testing. We performed a retrospective cohort analysis that compared outcomes between the preimplementation period (January-November 2018) and the postimplementation period (January-November 2019). Patients were tested for Treponema pallidum antibody (TPA) using a multiplex flow immunoassay (MFI), and positive results were confirmed by rapid plasma reagin (RPR). The primary implementation outcome was the number of syphilis tests/month, and the primary clinical outcome was the number of syphilis diagnoses/month (defined as positive TPA MFI and RPR). We performed an interrupted time-series analysis to evaluate the effect of implementing the alert over time. RESULTS: Four-hundred and ninety-four and 1106 unique patients were tested for syphilis in the preimplementation and postimplementation periods, respectively. Syphilis testing increased by 55.6 tests/month (95% CI 45.9 to 65.3, p<0.001) following alert implementation. Patients tested in the postimplementation period who were tested using the alert were much younger (difference: 14 years (95% CI 12 to 15)) and were more likely to be female (difference: 15% (95% CI 8 to 21)) and African-American (difference: 11% (95% CI 5 to 17)) than patients tested by clinician-initiated testing. Presumptive syphilis diagnoses increased from 3.4 diagnoses/month to 7.9 diagnoses/month (difference, 4.5 (95% CI 2.2 to 6.9), p<0.001). CONCLUSIONS: Our study demonstrates that use of a targeted EHR alert testing protocol can increase syphilis testing and diagnosis and may reduce clinician bias in testing.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5' untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3'UTR. Finally, 5 variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.

Interrupted time-series analysis to evaluate the impact of a behavioral change outpatient antibiotic stewardship intervention.

Objective: We evaluated the effect of a behaviorally enhanced quality improvement intervention in reducing the number of antibiotic prescriptions written for antibiotic nonresponsive acute respiratory infections (ARIs). A secondary objective was identifying whether a reduction in inappropriate antibiotic prescriptions, if present, persisted after the immediate implementation of the intervention. Design: Nonrandomized, quasi-experimental study conducted from January 2017 through February 2020. Setting: University of California, Davis Health outpatient clinics. In total, 21 pediatric, family, and internal medicine practices in 10 cities and towns were included. Patients: Patients evaluated by a participating physician at an enrolled practice site during the study period with diagnoses (primary and secondary) from the International Classification of Diseases, Tenth Revision codes consistent with antibiotic nonresponsive ARI diagnoses. Intervention: A behaviorally enhanced quality improvement intervention to reduce inappropriate prescribing for antibiotic nonresponsive ARI. Results: In total, 63,028 eligible patient visits across 21 locations were included in the analysis. The most frequently prescribed antibiotic for antibiotic nonresponsive ARI was azithromycin (n = 3,551), followed by amoxicillin (n = 924). Overall, the intervention was associated with an immediate 46% reduction in antibiotic prescriptions for antibiotic nonresponsive ARI (P = .001) following the intervention. We detected no significant change in the month-to-month trend after the intervention was implemented (P = .87), indicating that the reduction was sustained throughout the postintervention period. Conclusion: Our findings demonstrate that a behaviorally enhanced quality improvement intervention to reduce inappropriate prescribing for antibiotic nonresponsive ARI in ambulatory care encounters was successful in reducing potentially inappropriate prescriptions for presumed antibiotic nonresponsive ARI.

Unexpected specificity within dynamic transcriptional protein-protein complexes.

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator-not often considered as contributing to binding-play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity.

The immunorecognition, subcellular compartmentalization, and physicochemical properties of nucleic acid nanoparticles can be controlled by composition modification.

Nucleic acid nanoparticles (NANPs) have become powerful new platforms as therapeutic and diagnostic tools due to the innate biological ability of nucleic acids to identify target molecules or silence genes involved in disease pathways. However, the clinical application of NANPs has been limited by factors such as chemical instability, inefficient intracellular delivery, and the triggering of detrimental inflammatory responses following innate immune recognition of nucleic acids. Here, we have studied the effects of altering the chemical composition of a circumscribed panel of NANPs that share the same connectivity, shape, size, charge and sequences. We show that replacing RNA strands with either DNA or chemical analogs increases the enzymatic and thermodynamic stability of NANPs. Furthermore, we have found that such composition changes affect delivery efficiency and determine subcellular localization, effects that could permit the targeted delivery of NANP-based therapeutics and diagnostics. Importantly, we have determined that altering NANP composition can dictate the degree and mechanisms by which cell immune responses are initiated. While RNA NANPs trigger both TLR7 and RIG-I mediated cytokine and interferon production, DNA NANPs stimulate minimal immune activation. Importantly, incorporation of 2'F modifications abrogates RNA NANP activation of TLR7 but permits RIG-I dependent immune responses. Furthermore, 2'F modifications of DNA NANPs significantly enhances RIG-I mediated production of both proinflammatory cytokines and interferons. Collectively this indicates that off-target effects may be reduced and/or desirable immune responses evoked based upon NANPs modifications. Together, our studies show that NANP composition provides a simple way of controlling the immunostimulatory potential, and physicochemical and delivery characteristics, of such platforms.

Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5'UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3'UTR. Finally, five variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.

Innate immune responses triggered by nucleic acids inspire the design of immunomodulatory nucleic acid nanoparticles (NANPs).

The unknown immune stimulation by nucleic acid nanoparticles (NANPs) has become one of the major impediments to a broad spectrum of clinical developments of this novel technology. Having evolved to defend against bacterial and viral nucleic acids, mammalian cells have established patterns of recognition that are also the pathways through which NANPs can be processed. Explorations into the immune stimulation brought about by a vast diversity of known NANPs have shown that variations in design correlate with variations in immune response. Therefore, as the mechanisms of stimulation are further elucidated, these trends are now being taken into account in the design phase to allow for development of NANPs that are tailored for controlled immune activation or quiescence.

R-BIND: An Interactive Database for Exploring and Developing RNA-Targeted Chemical Probes.

While the opportunities available for targeting RNA with small molecules have been widely appreciated, the challenges associated with achieving specific RNA recognition in biological systems have hindered progress and prevented many researchers from entering the field. To facilitate the discovery of RNA-targeted chemical probes and their subsequent applications, we curated the RNA-targeted BIoactive ligaNd Database (R-BIND). This collection contains an array of information on reported chemical probes that target non-rRNA and have biological activity, and analysis has led to the discovery of RNA-privileged properties. Herein, we developed an online platform to make this information freely available to the community, offering search options, a suite of tools for probe development, and an updated R-BIND data set with detailed experimental information for each probe. We repeated the previous cheminformatics analysis on the updated R-BIND list and found that the distinguishing physicochemical, structural, and spatial properties remained unchanged, despite an almost 50% increase in the database size. Further, we developed several user-friendly tools, including queries based on cheminformatic parameters, experimental details, functional groups, and substructures. In addition, a nearest neighbor algorithm can assess the similarity of user-uploaded molecules to R-BIND ligands. These tools and resources can be used to design small molecule libraries, optimize lead ligands, or select targets, probes, assays, and control experiments. Chemical probes are critical to the study and discovery of novel functions for RNA, and we expect this resource to greatly assist researchers in exploring and developing successful RNA-targeted probes.