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.

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.

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.

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.

Insights into the development of chemical probes for RNA.

Over the past decade, the RNA revolution has revealed thousands of non-coding RNAs that are essential for cellular regulation and are misregulated in disease. While the development of methods and tools to study these RNAs has been challenging, the power and promise of small molecule chemical probes is increasingly recognized. To harness existing knowledge, we compiled a list of 116 ligands with reported activity against RNA targets in biological systems (R-BIND). In this survey, we examine the RNA targets, design and discovery strategies, and chemical probe characterization techniques of these ligands. We discuss the applicability of current tools to identify and evaluate RNA-targeted chemical probes, suggest criteria to assess the quality of RNA chemical probes and targets, and propose areas where new tools are particularly needed. We anticipate that this knowledge will expedite the discovery of RNA-targeted ligands and the next phase of the RNA revolution.

Instrument-assisted soft tissue mobilization and proprioceptive neuromuscular facilitation techniques improve hamstring flexibility better than static stretching alone: a randomized clinical trial.

Objectives: Tight hamstrings contribute to inefficiency of movement and increased risk for injury. Static stretching is the most common intervention for this problem, but the use of alternatives like instrument-assisted soft tissue mobilization (IASTM) and proprioceptive neuromuscular facilitation (PNF) is increasing among clinicians. This study examined two prospective studies with the common aim of demonstrating the effectiveness of IASTM or PNF over static stretching for improving hamstring tightness. Methods: Nondisabled adults were recruited on a university campus. IASTM study: N = 17 (11 males and 6 females). PNF study: N = 23 (7 males and 16 females). Hip flexion range of motion was measured with a passive straight leg raise (for IASTM) or active straight leg raise (for PNF) before and after stretching. Participants performed a self-static stretch on one leg and received the alternative intervention on the contralateral leg. The two studies were analyzed separately for reliability indices and significant differences between interventions. Results: Hip flexion measures showed good reliability in both studies (intraclass correlation coefficient = 0.97) with a minimal detectable change of <4.26. Both studies showed significant interactions between time and intervention (p < 0.05). Follow-up analyses revealed PNF and IASTM interventions resulted in greater increases in hip flexion range than static stretching. Discussion: These findings demonstrate the effectiveness of PNF and IASTM techniques over static stretching for hamstring flexibility. These interventions provide more efficient alternatives for improving flexibility in the clinic, allowing greater progress in a shorter period of time than an equivalent static stretching program. Level of Evidence: 1b.

Discovery of Small Molecule Ligands for MALAT1 by Tuning an RNA-Binding Scaffold.

Structural studies of the 3'-end of the oncogenic long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) confirmed a unique triple-helix structure. This structure enables accumulation of the transcript, and high levels of MALAT1 are found in several cancers. Here, we synthesize a small molecule library based on an RNA-binding scaffold, diphenylfuran (DPF), screen it against a variety of nucleic acid constructs, and demonstrate for the first time that the MALAT1 triple helix can be selectively targeted with small molecules. Computational analysis revealed a trend between subunit positioning and composition on DPF shape and intramolecular interactions, which in turn generally correlated with selectivity and binding strengths. This work thus provides design strategies toward chemical probe development for the MALAT1 triple helix and suggests that comprehensive analyses of RNA-focused libraries can generate insights into selective RNA recognition.

Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients.

Programmable Nucleic Acid Based Polygons with Controlled Neuroimmunomodulatory Properties for Predictive QSAR Modeling.

In the past few years, the study of therapeutic RNA nanotechnology has expanded tremendously to encompass a large group of interdisciplinary sciences. It is now evident that rationally designed programmable RNA nanostructures offer unique advantages in addressing contemporary therapeutic challenges such as distinguishing target cell types and ameliorating disease. However, to maximize the therapeutic benefit of these nanostructures, it is essential to understand the immunostimulatory aptitude of such tools and identify potential complications. This paper presents a set of 16 nanoparticle platforms that are highly configurable. These novel nucleic acid based polygonal platforms are programmed for controllable self-assembly from RNA and/or DNA strands via canonical Watson-Crick interactions. It is demonstrated that the immunostimulatory properties of these particular designs can be tuned to elicit the desired immune response or lack thereof. To advance the current understanding of the nanoparticle properties that contribute to the observed immunomodulatory activity and establish corresponding designing principles, quantitative structure-activity relationship modeling is conducted. The results demonstrate that molecular weight, together with melting temperature and half-life, strongly predicts the observed immunomodulatory activity. This framework provides the fundamental guidelines necessary for the development of a new library of nanoparticles with predictable immunomodulatory activity.

Discovery of Key Physicochemical, Structural, and Spatial Properties of RNA-Targeted Bioactive Ligands.

While a myriad non-coding RNAs are known to be essential in cellular processes and misregulated in diseases, the development of RNA-targeted small molecule probes has met with limited success. To elucidate the guiding principles for selective small molecule/RNA recognition, we analyzed cheminformatic and shape-based descriptors for 104 RNA-targeted ligands with demonstrated biological activity (RNA-targeted BIoactive ligaNd Database, R-BIND). We then compared R-BIND to both FDA-approved small molecule drugs and RNA ligands without reported bioactivity. Several striking trends emerged for bioactive RNA ligands, including: 1) Compliance to medicinal chemistry rules, 2) distinctive structural features, and 3) enrichment in rod-like shapes over others. This work provides unique insights that directly facilitate the selection and synthesis of RNA-targeted libraries with the goal of efficiently identifying selective small molecule ligands for therapeutically relevant RNAs.

Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d.

A member of the TTP family of proteins, TIS11d binds RNA with high specificity using a pair of CCCH-type tandem zinc fingers separated by a 18 residue long linker. Our previous work showed that the formation of hydrogen bonds between the C-terminal residue E220 and the residues of the linker region stabilized a compact structure of TIS11d in the absence of RNA. To investigate the role of the C-terminal residues in the structure of unbound TIS11d, the E220A mutant and the truncation mutant lacking the last two residues (D219/E220) were studied using molecular dynamics, NMR spectroscopy, and biochemical methods. This study confirmed the importance of the charged residues D219 and E220 in maintaining structural stability in unbound TIS11d and elucidated the underlying physical mechanisms. We observed a greater structural heterogeneity for the residues of the linker in the molecular dynamics trajectories of both mutant proteins relative to the wild-type. This heterogeneity was more pronounced in the D219/E220 deletion mutant than in the E220A mutant, indicating that a greater reduction of the charge of the C-terminus results in greater flexibility. In agreement with the increased flexibility and the reduced number of negatively charged residues of the D219/E220 deletion mutant, we measured more unfavorable entropic and a more favorable enthalpic contribution to the free energy of RNA binding in the mutant than in the wild-type protein. The relative orientation of the zinc fingers was stabilized by the electrostatic interaction between E220 and positively charged residues of the linker in TIS11d. In the E220A mutant, the relative orientation of the zinc fingers was less constrained, whereas in the D219/E220 deletion mutant, little orientational preference was observed. We posit that favorable electrostatic interactions provide a mechanism to promote preferential orientation of separate domains without imposing structural rigidity.

KDM1 class flavin-dependent protein lysine demethylases.

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1-selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

Defining the role of TORC1/2 in multiple myeloma.

Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.

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.

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.

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.