Titin is a nucleolar protein in neurons.

Titin is the largest protein produced by living cells and its function as a molecular spring in striated muscle is well characterized (1, 2). Here we demonstrate that titin isoforms in the same size range as found in muscle are prominent neuronal proteins in both the central and peripheral nervous systems, including motor neurons in the spinal cord and brain. Within these neurons, titin localizes to the dense fibrillar component of the nucleolus, the site of ribosomal RNA biogenesis and modification, and a critical site of dysfunction in neurodegenerative disease (3-5). Additionally, we show that the levels of both titin mRNA and protein are altered in the spinal cord of SOD1G93A mice, a commonly used model of amyotrophic lateral sclerosis, indicating that titin mediated nucleolar events may in fact contribute to the pathobiology of disease.

A different approach to teaching pre-clerkship students physical diagnosis: standardized patient instructor-senior medical student teaching teams.

BACKGROUND: Faculty have traditionally taught the physical examination (PE) to novice medical students (pre-clerkship students.), despite recruiting and cost issues and problems standardizing their approach. ACTIVITY: We present a model using standardized patient instructor (SPI)-fourth year medical student (MS4) teams to teach PE to pre-clerkship students, leveraging the benefits of co-teaching and peer-assisted learning. RESULTS: Surveys of pre-clerkship students, MS4s and SPIs indicate positive perceptions of the program, including MS4s reporting significant growth in their professional identities as educators. Pre-clerkship students' performance on the spring clinical skills exams was equivalent to or better than their peer performance pre-program implementation. IMPLICATIONS: SPI-MS4 teams can effectively teach novice students the mechanics and clinical context of the beginners' physical exam.

ParSe 2.0: A web tool to identify drivers of protein phase separation at the proteome level.

We have developed an algorithm, ParSe, which accurately identifies from the primary sequence those protein regions likely to exhibit physiological phase separation behavior. Originally, ParSe was designed to test the hypothesis that, for flexible proteins, phase separation potential is correlated to hydrodynamic size. While our results were consistent with that idea, we also found that many different descriptors could successfully differentiate between three classes of protein regions: folded, intrinsically disordered, and phase-separating intrinsically disordered. Consequently, numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. Built from that finding, ParSe 2.0 uses an optimal set of property scales to predict domain-level organization and compute a sequence-based prediction of phase separation potential. The algorithm is fast enough to scan the whole of the human proteome in minutes on a single computer and is equally or more accurate than other published predictors in identifying proteins and regions within proteins that drive phase separation. Here, we describe a web application for ParSe 2.0 that may be accessed through a browser by visiting https://stevewhitten.github.io/Parse_v2_FASTA to quickly identify phase-separating proteins within large sequence sets, or by visiting https://stevewhitten.github.io/Parse_v2_web to evaluate individual protein sequences.

Intrinsically disordered regions that drive phase separation form a robustly distinct protein class.

Protein phase separation is thought to be a primary driving force for the formation of membrane-less organelles, which control a wide range of biological functions from stress response to ribosome biogenesis. Among phase-separating (PS) proteins, many have intrinsically disordered regions (IDRs) that are needed for phase separation to occur. Accurate identification of IDRs that drive phase separation is important for testing the underlying mechanisms of phase separation, identifying biological processes that rely on phase separation, and designing sequences that modulate phase separation. To identify IDRs that drive phase separation, we first curated datasets of folded, ID, and PS ID sequences. We then used these sequence sets to examine how broadly existing amino acid property scales can be used to distinguish between the three classes of protein regions. We found that there are robust property differences between the classes and, consequently, that numerous combinations of amino acid property scales can be used to make robust predictions of protein phase separation. This result indicates that multiple, redundant mechanisms contribute to the formation of phase-separated droplets from IDRs. The top-performing scales were used to further optimize our previously developed predictor of PS IDRs, ParSe. We then modified ParSe to account for interactions between amino acids and obtained reasonable predictive power for mutations that have been designed to test the role of amino acid interactions in driving protein phase separation. Collectively, our findings provide further insight into the classification of IDRs and the elements involved in protein phase separation.

Peer-Led Focus Groups Identify Barriers to Healthy Lifestyle in African American Adolescents from Baltimore City.

Objectives: Black youth are disproportionately affected by the US obesity epidemic. Early-age obesity often continues into adulthood and is associated with a higher risk of diabetes, cardiovascular disease, and premature death. Few studies have incorporated community-based participatory research (CBPR) facilitated by youth to provide frank discussions among teens living in inner cities about challenges and facilitators in maintaining a healthy weight and to design teen-identified interventions. Design: Black youth (n=10) were recruited from a Baltimore City high school during the 2019 to 2020 academic year and were trained by seasoned investigators and mentored by graduate and undergraduate students on qualitative methods using CBPR. These youth then implemented focus groups with their peers aged 15 to 18 years (10 focus groups of 10 teens each). Topics included healthy lifestyle knowledge, behaviors, attitudes, and suggested interventions. Content analyses were conducted using dual-rater techniques. Results: Focus group themes yielded strengths and challenges of weight maintenance for Black youth at various levels, including in their personal lives, families, school, and community. Themes also suggested several technology-based possibilities using social media to reach Black youth about healthy living practices. Conclusions: Engagement of Black youth in CBPR projects can yield valuable data to design more culturally responsive and developmentally appropriate interventions. Youth are competent collectors of information to identify needed changes in their schools/communities and about the use of technology/social media to facilitate improved health practices among their peers and should be involved early in the process of developing targeted obesity prevention interventions and/or programs.

An educational intervention to facilitate appropriate subspecialty referrals: a study assessing resident communication skills.

BACKGROUND: Our goal was to improve pediatric residents' advanced communication skills in the setting of referral to address the entrustable professional activity of subspecialty referral identified by the American Board of Pediatrics. To accomplish this aim, we created a referral and consultation curriculum to teach and assess core communication skills in subspecialty referral involving an adolescent with syncope, an anxiety-provoking symptom that is rarely associated with serious pathology. METHODS: We utilized blended multimodal educational interventions to improve resident communication skills in referral of patients. Trainees participated in 1) an interactive online module on syncope focusing on "red-flag" symptoms that would warrant a subspecialty cardiology referral and 2) a 4-h intervention with Standardized Parents (SPs), focusing on the case-based application of communication skills. Communication skills were assessed by two pre- and post- Objective Structured Clinical Examination encounters of patients with syncope, with an SP evaluation using a 20-item checklist. Analysis was performed with Sign test and McNemar's test. Trainees provided feedback on a Critical Incident Questionnaire, which was analyzed qualitatively. RESULTS: Sixty-four residents participated. There was an overall improvement in communication skills based on SP scores (82.7 ± 10.9% to 91.7 ± 5.0%, p < 0.001), and 13/20 items demonstrated significant improvement post-intervention. Residents' improved performance enabled them to address patient/family emotions, explain referral logistics, and clarify concerns to agree on a plan. CONCLUSIONS: By participating in this curriculum, residents' communication skills improved immediately post-intervention. Further research is needed to assess if this intervention improves patient care by providing residents with enduring skills to judiciously manage the referral process.

Supporting Zoo Asian Elephant (Elephas maximus) Welfare and Herd Dynamics with a More Complex and Expanded Habitat.

Ensuring good health and welfare is an increasingly important consideration for conservation of endangered species, whether free-ranging or managed to varying degrees under human care. The welfare-based design of a new habitat for Asian elephants at the Oregon Zoo focused on meeting the elephants' physical, physiological, psychological, and social needs 24 h a day and across life stages. The habitat was designed to encourage activity, promote species-typical behaviors, support changing social dynamics, offer increased opportunities for choice, and provide biologically meaningful challenges. In this 4-year study, we monitored elephant health and welfare indicators throughout the transition and acclimation from the previous habitat to the new habitat. Several welfare indicators obtained through longitudinal hormone analyses, behavior assessments, and GPS measurement of walking distance and space use provided evidence that these goals were achieved. The elephants were more active and walked farther on a daily basis in the new habitat, with an average walking distance of over 15 km per day. A switch from primarily caretaker-delivered food to seeking food on their own indicates that the disbursement of food with less temporal and spatial predictability increased foraging opportunities, which better satisfies appetitive motivations important for psychological well-being. All individuals showed adaptive and normal adrenal responses to change and challenge, with the highest fecal glucocorticoid metabolite (FGM) concentrations and variability during the construction phase, and a return to previous baseline concentrations in the new habitat, suggesting they acclimated well to the new environment. The elephants expressed a diverse range of species-typical behaviors and demonstrated social dynamics of a healthy herd in both habitats with transitions of individuals through life stages. They exhibited more autonomy in choosing whom to associate with socially and also by choosing different aspects of their environment with regular indoor/outdoor access and extensive resource use in the new habitat. Findings indicate that the complexity and flexibility of the new habitat and habitat management has been effective in improving overall welfare by providing meaningful challenges and the opportunity to express appetitive behaviors, by offering choice in environmental conditions, and by providing the space and resource distribution to support evolving herd dynamics and increased social equity for individuals.

Structural and Energetic Characterization of the Denatured State from the Perspectives of Peptides, the Coil Library, and Intrinsically Disordered Proteins.

The α and polyproline II (PPII) basins are the two most populated regions of the Ramachandran map when constructed from the protein coil library, a widely used denatured state model built from the segments of irregular structure found in the Protein Data Bank. This indicates the α and PPII conformations are dominant components of the ensembles of denatured structures that exist in solution for biological proteins, an observation supported in part by structural studies of short, and thus unfolded, peptides. Although intrinsic conformational propensities have been determined experimentally for the common amino acids in short peptides, and estimated from surveys of the protein coil library, the ability of these intrinsic conformational propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs), an increasingly important class of proteins in cell function, has thus far proven elusive to establish. Recently, we demonstrated that the sequence dependence of the mean hydrodynamic size of IDPs in water and the impact of heat on the coil dimensions, provide access to both the sequence dependence and thermodynamic energies that are associated with biases for the α and PPII backbone conformations. Here, we compare results from peptide-based studies of intrinsic conformational propensities and surveys of the protein coil library to those of the sequence-based analysis of heat effects on IDP hydrodynamic size, showing that a common structural and thermodynamic description of the protein denatured state is obtained.

How Do Standardized Patients Form Their Complex Identities? The Impact of Interactions With Medical Students.

INTRODUCTION: Little is known about the nature of standardized patient (SP) identity and its formation. Understanding identity and formation can have important implications for recruiting, working with, and retaining SPs. Social relational theory explains how identity is formed in a social context in relationship with others. In keeping with social relational theory, this study explored SP identity formation through the SP experience with one of the most important elements of their social context-medical students. METHODS: Using qualitative methods, we conducted four 1-hour semistructured focus group interviews with SPs (N = 18). Interviews were audio recorded and transcribed verbatim. We used a constant comparative approach and consensus development to analyze the data and identify codes, categories, and ultimately themes. We used multiple methods to ensure credibility and trustworthiness of the study, as well as transparency of methods and thick rich descriptions to enable readers to judge transferability of the findings and conclusions. RESULTS: We identified 4 major themes. Through their interactions, SPs experienced: (1) identity transformation toward a new professional identity; (2) self-actualization toward their maximum potential; (3) judgmental reactions to medical student behaviors; and (4) simulation-reality interaction between their simulated and real selves. CONCLUSIONS: Through their interactions with medical students, SPs experienced a transformation into a complex identity composed of multiple roles. Within this identity, they found purpose, fulfillment, and self-actualization through fostering students', as well as their own, personal, and professional growth and through developing a higher sense of social responsibility. Within this identity, they also encountered the challenges of navigating between judging and supporting medical students, and toggling between their real and simulated selves. Guided by the SPs' perspectives, and in conjunction with professional organizational standards, we provide recommendations for training, recruitment, and retention of SPs.

The La-related proteins: structures and interactions of a versatile superfamily of RNA-binding proteins.

The La-related proteins (LaRPs) are an ancient superfamily of RNA-binding proteins orchestrating the major fates of RNA, from processing and maturation to regulation of mRNA translation. LaRPs are instrumental in modulating complex assemblies where the RNA is bound, folded, processed, escorted and presented to the functional effectors often through recruitment of protein partners. This intricate web of protein-RNA and protein-protein interactions is enabled by the modular nature of the LaRPs, comprising several structured domains connected by flexible linkers, and other sequences lacking recognizable folded motifs. Recent structures, together with biochemical and biophysical studies, have provided insights into how each LaRP family has evolved unique mechanisms of RNA recognition, not only through the conserved RNA-binding unit, the La-module, but also mediated by other family-specific motifs. Furthermore, in a series of unexpected twists and turns, they have revealed that the dynamic and conformational interplay of multi-structured domains and disordered regions operate in unison to achieve RNA substrate discrimination. This review proposes a perspective of our current knowledge of the structure-function relationship of the LaRP superfamily.

Oral Health of Older Adults in Washington State.

Purpose: An aging population, combined with increasing tooth retention, could significantly impact the dental care delivery system. The purpose of this study was to assess self-reported oral health and the factors associated with oral health outcomes among a random sample of older adults in Washington State.Methods: A telephone survey of adults 55+ years was used to collect information on factors associated with oral health, plus four outcome variables; substantial tooth loss (6+ teeth lost), oral problems, oral pain, and poor health of teeth. Data were weighted to reflect the state's age and gender statistics.Results: A total of 2,988 older adults completed the survey during 2017. Substantial tooth loss (18%), oral problems (17%) and oral pain (13%) were the most frequently reported issues. Of the adults with teeth, 17% reported fair/poor health of teeth. Compared to adults with an income of $75,000 or more, adults with an income less than $25,000 were twice as likely to have substantial tooth loss and oral problems (OR=2.1 and 2.2, respectively) and were three times more likely to report oral pain and poor health of teeth (OR=3.1 and 3.3, respectively). The oldest old (adults 75+ years), as compared to those 55-64 years, were significantly more likely to have substantial tooth loss (OR=2.6) but were less likely to report oral problems (OR=0.6), pain (OR=0.3), or poor health of teeth (OR=0.5).Conclusions: Although the majority of Washington's older adults report having good oral health, a small subgroup has oral problems which may have a negative impact on quality of life.

An island of wildlife in a human-dominated landscape: The last fragment of primary forest on the Osa Peninsula's Golfo Dulce coastline, Costa Rica.

Habitat loss and fragmentation, together with related edge effects, are the primary cause of global biodiversity decline. Despite a large amount of research quantifying and demonstrating the degree of these effects, particularly in top predators and their prey, most fragmented patches are lost before their conservation value is recognized. This study evaluates terrestrial vertebrates in Playa Sandalo, in the Osa Peninsula of Costa Rica, which represents the last patch of "primary" forest in the most developed part of this region. Our study indicates that the diversity of ground species detected within Playa Sandalo rival other areas under active conservation like Lapa Rios Ecolodge. Historical fragmentation, together with the maintenance of forest cover in isolated conditions, are potentially responsible for the species composition observed within Playa Sandalo; facilitating the development of a prey-predator system including ocelots, medium-size mammals, and birds at the top of the trophic chain. The high diversity of both habitat and vertebrates, its prime location and cultural value, as well as its unique marine importance represent the ideal conditions for conservation. Conservation of Playa Sandalo, and other small tropical forest remnants, might represent the only management option for wildlife conservation within ever growing human-dominated landscapes.

Implementing Yoga Into the Management of Patients With Refractory Low Back Pain in an Outpatient Clinic Setting.

Purpose: To evaluate the effectiveness of implementing yoga into the treatment of patients with chronic low back pain. Design: Quantitative analysis with opportunity for qualitative feedback. Method: Effectiveness of this complementary treatment was assessed using a pretest/posttest design of patients who volunteered to participate in yoga classes as part of their back pain management. Measurements included low back pain rating, perception of back pain interference with daily activities, and self-efficacy in dealing with chronic low back pain. Findings: Although no statistically significant findings were found due to the small sample size, most participants demonstrated improved individual scores on all measurement surveys including qualitative comments. Conclusion: Based on the findings of this pilot study, further studies on implementing yoga into the treatment of chronic low back pain are encouraged.

Measuring Low-Picomolar Apparent Binding Affinities by Minigel Electrophoretic Mobility Shift.

Measuring protein/DNA interactions that have apparent binding affinity constants in the low-picomolar range presents a unique experimental challenge. To probe the sequence specificity of telomere binding proteins, our laboratory has developed an electrophoretic mobility shift assay protocol that allows for the routine measurement of K D,app values in the 1-20 pM range. Here, we describe the protocol and highlight the particular considerations that should be made to successfully and reproducibly measure high-affinity interactions between proteins and single-stranded DNA.

Horizontal Agarose Gel Mobility Shift Assay for Protein-RNA Complexes.

Recent advances in agarose gel electrophoresis protocols established conditions for the high-resolution separation of DNA and RNA using higher voltages combined with short run times. We subsequently developed a protocol for using these conditions to measure the binding affinity of a protein for an RNA ligand on an agarose gel. This native gel mobility shift assay is highly accessible, using common molecular biology reagents found in most laboratories. Here, we describe the protocol for carrying out native agarose gel electrophoresis to characterize the binding affinity of a protein for an RNA ligand. The electrophoresis time is less than 10 min, which minimizes the dissociation of protein and ligand. We have used the p19 siRNA binding protein and its cognate dsRNA ligand to demonstrate strategies for identifying optimal conditions to measure apparent binding constants using this agarose gel shift system.

Evaluating the use of high-fidelity simulators during mock neonatal resuscitation scenarios in trying to improve confidence in residents.

OBJECTIVE: The purpose of this study was to investigate the effect of the use of high-fidelity simulators with multidisciplinary teaching on self-reported confidence in residents. METHODS: A total of 26 residents participated in a session led by a pediatric faculty member and a Neonatal Intensive Care Unit transport nurse using a high-fidelity pediatric simulator. Multiple scenarios were presented and each resident took turns in various roles. Pre-intervention surveys based on a 5-point Likert-type scale were given before the scenarios and were compared to the results of identical post-intervention surveys. RESULTS: Statistically significant (p < 0.05) pre to post gains for self-confidence were observed. Improvements in confidence were analyzed using the mean difference. The largest improvement in confidence was seen in the ability to treat neonatal respiratory arrest. This was followed by the ability to supervise/run a code, and the ability to place an umbilical venous catheter. CONCLUSION: These results revealed that high-fidelity simulation-based training has significant positive gains in residents' self-reported confidence.

A novel variant in TAF1 affects gene expression and is associated with X-linked TAF1 intellectual disability syndrome.

We investigated the genome of a 5-year-old male who presented with global developmental delay (motor, cognitive, and speech), hypotonia, possibly ataxia, and cerebellar hypoplasia of unknown origin. Whole genome sequencing (WGS) and mRNA sequencing (RNA-seq) were performed on a family having an affected proband, his unaffected parents, and maternal grandfather. To explore the molecular and functional consequences of the variant, we performed cell proliferation assays, quantitative real-time PCR (qRT-PCR) array, immunoblotting, calcium imaging, and neurite outgrowth experiments in SH-SY5Y neuroblastoma cells to compare the properties of the wild-type TATA-box-binding protein factor 1 (TAF1), deletion of TAF1, and TAF1 variant p.Ser1600Gly samples. The whole genome data identified several gene variants. However, the genome sequence data failed to implicate a candidate gene as many of the variants were of unknown significance. By combining genome sequence data with transcriptomic data, a probable candidate variant, p.Ser1600Gly, emerged in TAF1. Moreover, the RNA-seq data revealed a 90:10 extremely skewed X-chromosome inactivation (XCI) in the mother. Our results showed that neuronal ion channel genes were differentially expressed between TAF1 deletion and TAF1 variant p.Ser1600Gly cells, when compared with their respective controls, and that the TAF1 variant may impair neuronal differentiation and cell proliferation. Taken together, our data suggest that this novel variant in TAF1 plays a key role in the development of a recently described X-linked syndrome, TAF1 intellectual disability syndrome, and further extends our knowledge of a potential link between TAF1 deficiency and defects in neuronal cell function.

Analysis of common barriers to rural patients utilizing hospice and palliative care services: An integrated literature review.

BACKGROUND AND PURPOSE: The aim of this literature review is to explore barriers and potential solutions related to hospice (HC) and palliative care (PC) services among rural residents. Although the healthcare system is continually advancing, healthcare providers may not be optimizing HC and PC referrals for the growing rural population who underutilize these services. Suggested methods to close the utilization gap between HC and PC services among rural patients appear feasible, but universal effectiveness cannot be determined. METHODS: An integrative literature review was conducted to evaluate diverse sources of literature. An electronic literature search was carried out using databases MEDLINE, CINAHL, Cochrane, PsycINFO, and Pubmed. The search was limited to English only, full text, peer reviewed, and published between 2010 and 2016. Search terms included rural, hospice, palliative, care access, and barriers. CONCLUSION: There are several barriers that interrelate to decreased utilization of PC and HC for rural populations and there are many options for overcoming them to equalize care. IMPLICATIONS FOR PRACTICE: Although advances to the general healthcare system are expediently rising, the rural patient population seems to fall short of these important life-changing services, especially in the realm of PC/HC. Beginning in primary care, this patient population can be affected and included in a positive manner.

Recombinant expression and purification of the RNA-binding LARP6 proteins from fish genetic model organisms.

The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components. The domain topologies of the purified fish proteins were probed with limited proteolysis. The fish proteins contain an internal, protease-resistant 40 kDa domain, which is considerably more stable than the comparable domain from the human LARP6 protein. The fish proteins are therefore a lucrative model system in which to study both the evolutionary divergence of this family of La-related proteins and the structure and conformational dynamics of the domains that comprise the LARP6 protein.