Systematic exploration of dynamic splicing networks reveals conserved multistage regulators of neurogenesis.

Alternative splicing (AS) is a critical regulatory layer; yet, factors controlling functionally coordinated splicing programs during developmental transitions are poorly understood. Here, we employ a screening strategy to identify factors controlling dynamic splicing events important for mammalian neurogenesis. Among previously unknown regulators, Rbm38 acts widely to negatively control neural AS, in part through interactions mediated by the established repressor of splicing, Ptbp1. Puf60, a ubiquitous factor, is surprisingly found to promote neural splicing patterns. This activity requires a conserved, neural-differential exon that remodels Puf60 co-factor interactions. Ablation of this exon rewires distinct AS networks in embryonic stem cells and at different stages of mouse neurogenesis. Single-cell transcriptome analyses further reveal distinct roles for Rbm38 and Puf60 isoforms in establishing neuronal identity. Our results describe important roles for previously unknown regulators of neurogenesis and establish how an alternative exon in a widely expressed splicing factor orchestrates temporal control over cell differentiation.

Efficient and Accurate Quantitative Profiling of Alternative Splicing Patterns of Any Complexity on a Laptop.

Alternative splicing (AS) is a widespread process underlying the generation of transcriptomic and proteomic diversity and is frequently misregulated in human disease. Accordingly, an important goal of biomedical research is the development of tools capable of comprehensively, accurately, and efficiently profiling AS. Here, we describe Whippet, an easy-to-use RNA-seq analysis method that rapidly-with hardware requirements compatible with a laptop-models and quantifies AS events of any complexity without loss of accuracy. Using an entropic measure of splicing complexity, Whippet reveals that one-third of human protein coding genes produce transcripts with complex AS events involving co-expression of two or more principal splice isoforms. We observe that high-entropy AS events are more prevalent in tumor relative to matched normal tissues and correlate with increased expression of proto-oncogenic splicing factors. Whippet thus affords the rapid and accurate analysis of AS events of any complexity, and as such will facilitate future biomedical research.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism. Approximately 200 genes associated with functionally diverse regulatory layers and enriched in genetic links to autism control neuronal microexons. Remarkably, the widely expressed RNA binding proteins Srsf11 and Rnps1 directly, preferentially, and frequently co-activate these microexons. These factors form critical interactions with the neuronal splicing regulator Srrm4 and a bi-partite intronic splicing enhancer element to promote spliceosome formation. Our study thus presents a versatile system for the identification of entire splicing regulatory pathways and further reveals a common mechanism for the definition of neuronal microexons that is disrupted in autism.

Effectively teaching cultural competence in a pre-professional healthcare curriculum.

BACKGROUND: There has been research documenting the rising numbers of racial and ethnic minority groups in the United States. With this rise, there is increasing concern over the health disparities that often affect these populations. Attention has turned to how clinicians can improve health outcomes and how the need exists to educate healthcare professionals on the practice of cultural competence. Here we present one successful approach for teaching cultural competence in the healthcare curriculum with the development of an educational session on cultural competence consisting of case-based, role-play exercises, class group discussions, online discussion boards, and a lecture PowerPoint presentation. METHODS: Cultural competence sessions were delivered in a pre-dental master's program to 178 students between 2017 and 2020. From 2017 to 2019, the sessions were implemented as in-person, case-based, role-play exercises. In 2020, due to in-person limitations caused by the COVID-19 pandemic, students were asked to read the role-play cases and provide a reflection response using the online Blackboard Learn discussion board platform. Evaluation of each session was performed using post-session survey data. RESULTS: Self-reported results from 2017 to 2020 revealed that the role-play exercises improved participant's understanding of components of cultural competence such as communication in patient encounters (95%), building rapport with patients (94%), improving patient interview skills (95%), and recognition of students own cultural biases when working with patients (93%). CONCLUSIONS: Students were able to expand their cultural awareness and humility after completion of both iterations of the course session from 2017 to 2019 and 2020. This session can be an effective method for training healthcare professionals on cultural competence.

Total Energy Expenditure and Nutritional Intake in Continuous Multiday Ultramarathon Events.

Continuous multiday ultramarathon competitions are increasingly popular and impose extreme energetic and nutritional demands on competitors. However, few data have been published on energy expenditure during these events. Here, we report doubly labeled water-derived measures of total energy expenditure (in kilocalories per day) and estimated physical activity level (PAL: total energy expenditure/basal metabolic rate) collected from five elite and subelite finishers (four males and one female, age 34.6 ± 4.9 years)-and nutritional intake data from the winner-of the Cocodona 250, a ∼402-km race in Arizona, and from a fastest-known-time record (one male, age 30 years) on the ∼1,315-km Arizona Trail. PAL during these events exceeded four times basal metabolic rate (Cocodona range: 4.34-6.94; Arizona Trail: 5.63). Combining the results with other doubly labeled water-derived total energy expenditure data from ultraendurance events show a strong inverse relationship between event duration and PAL (r2 = .68, p < .0001). Cocodona race duration was inversely, though not significantly, associated with PAL (r2 = .70, p = .08). Water turnover varied widely between athletes and was not explained by PAL or body mass. The Cocodona race winner met ∼53% of energy demand via dietary intake, 85.6% of which was carbohydrate, while ∼47% of energy demand was met via catabolism of body energy stores. Together, these results illustrate the energetic deficits incurred during competitive continuous multiday ultramarathon efforts and implicate macronutrient absorption and/or storage as key factors in ultramarathon performance.

Why does strength training improve endurance performance?

OBJECTIVE: The specificity of training principle holds that adaptations to exercise training closely match capacity to the specific demands of the stimulus. Improvements in endurance sport performance gained through strength training are a notable exception to this principle. While the proximate mechanisms for how strength training produces muscular adaptations beneficial to endurance sports are increasingly well understood, the ultimate causes of this phenomenon remain unexplored. METHODS: Using a holistic approach tying together exercise physiology and evolution, I argue that we can reconcile the apparent "endurance training specificity paradox." RESULTS AND CONCLUSIONS: Competing selective pressures, inherited mammalian biology, and millennia of living in energy-scarce environments constrained our evolution as endurance athletes, but also imparted high muscular plasticity which can be exploited to improve endurance performance beyond what was useful in our evolutionary past.

Diversity and evolution of human eccrine sweat gland density.

The human eccrine sweat gland is central to the evolution of the human genus, permitting an enormous thermoregulatory sweating capacity that was essential to the human niche of high physical activity in open, hot, semi-arid environments. Despite a century of research inventorying the structure and function of eccrine glands and the physiological responses of human heat acclimation, we do not have a clear understanding of how intraspecific differences in eccrine density affect thermoregulation. Similarly, existing data does not comprehensively catalogue modern human diversity in this trait, nor do we understand the relative influences of evolutionary forces and phenotypic plasticity in shaping this diversity.

The evolution of eccrine sweat glands in human and nonhuman primates.

Sweating is an unusual thermoregulatory strategy for most mammals, yet is critical for humans. This trait is commonly hypothesized to result from human ancestors moving from a forest to a warmer and drier open environment. As soft tissue traits do not typically fossilize, this idea has been difficult to test. Therefore, we used a comparative approach to examine 15 eccrine gland traits from 35 primate species. For each trait we measured phylogenetic signal, tested three evolutionary models to explain trait variation, and used phylogenetic models to examine how traits varied in response to climate variables. Phylogenetic signal in traits varied substantially, with the two traits exhibiting the highest values being gland distribution on the body and percent eccrine vs. apocrine glands on the body. Variation in most traits was best explained by an Ornstein-Uhlenbeck model suggesting the importance of natural selection. Two traits were strongly predicted by climate. First, species with high eccrine gland glycogen content were associated with habitats exhibiting warm temperatures and low rainfall. Second, species with increased capillarization were associated with high temperature. Glycogen is a primary energy substrate powering sweat production and sodium reabsorption in the eccrine gland, and increased capillarization permits greater oxygen, glucose and electrolyte delivery. Thus, our results are evidence of natural selection for increased sweating capacity in primate species with body surface eccrine glands living in hot and dry climates. We suggest that selection for increased glycogen content and capillarization may have been part of initial increases in hominin thermoregulatory sweating capacity.

What did Hadropithecus eat, and why should paleoanthropologists care?

Over 40 years ago, Clifford Jolly noted different ways in which Hadropithecus stenognathus converged in its craniodental anatomy with basal hominins and with geladas. The Malagasy subfossil lemur Hadropithecus departs from its sister taxon, Archaeolemur, in that it displays comparatively large molars, reduced incisors and canines, a shortened rostrum, and thickened mandibular corpus. Its molars, however, look nothing like those of basal hominins; rather, they much more closely resemble molars of grazers such as Theropithecus. A number of tools have been used to interpret these traits, including dental microwear and texture analysis, molar internal and external morphology, and finite element analysis of crania. These tools, however, have failed to provide support for a simple dietary interpretation; whereas there is some consistency in the inferences they support, dietary inferences (e.g., that it was graminivorous, or that it specialized on hard objects) have been downright contradictory. Cranial shape may correlate poorly with diet. But a fundamental question remains unresolved: why do the various cranial and dental convergences exemplified by Hadropithecus, basal hominins, and Theropithecus exist? In this paper we review prior hypotheses regarding the diet of Hadropithecus. We then use stable carbon and nitrogen isotope data to elucidate this species' diet, summarizing earlier stable isotope analyses and presenting new data for lemurs from the central highlands of Madagascar, where Hadropithecus exhibits an isotopic signature strikingly different from that seen in other parts of the island. We offer a dietary explanation for these differences. Hadropithecus likely specialized neither on grasses nor hard objects; its staples were probably the succulent leaves of CAM plants. Nevertheless, aspects of prior hypotheses regarding the ecological significance of its morphology can be supported. Am. J. Primatol. 78:1098-1112, 2016. © 2015 Wiley Periodicals, Inc.

Tra2 protein biology and mechanisms of splicing control.

Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2ß in development.

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2ß (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2ß is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2ß binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2ß regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2ß binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2ß protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2ß. Versions of Tra2ß lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2ß protein.

Use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies.

IMPORTANCE: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE: To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS: Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.

Limits of Ultra: Towards an Interdisciplinary Understanding of Ultra-Endurance Running Performance.

Ultra-endurance running (UER) poses extreme mental and physical challenges that present many barriers to completion, let alone performance. Despite these challenges, participation in UER events continues to increase. With the relative paucity of research into UER training and racing compared with traditional endurance running distance (e.g., marathon), it follows that there are sizable improvements still to be made in UER if the limitations of the sport are sufficiently understood. The purpose of this review is to summarise our current understanding of the major limitations in UER. We begin with an evolutionary perspective that provides the critical background for understanding how our capacities, abilities and limitations have come to be. Although we show that humans display evolutionary adaptations that may bestow an advantage for covering large distances on a daily basis, these often far exceed the levels of our ancestors, which exposes relative limitations. From that framework, we explore the physiological and psychological systems required for running UER events. In each system, the factors that limit performance are highlighted and some guidance for practitioners and future research are shared. Examined systems include thermoregulation, oxygen delivery and utilisation, running economy and biomechanics, fatigue, the digestive system, nutritional and psychological strategies. We show that minimising the cost of running, damage to lower limb tissue and muscle fatigability may become crucial in UER events. Maintaining a sustainable core body temperature is critical to performance, and an even pacing strategy, strategic heat acclimation and individually calculated hydration all contribute to sustained performance. Gastrointestinal issues affect almost every UER participant and can be due to a variety of factors. We present nutritional strategies for different event lengths and types, such as personalised and evidence-based approaches for varying types of carbohydrate, protein and fat intake in fluid or solid form, and how to avoid flavour fatigue. Psychology plays a vital role in UER performance, and we highlight the need to be able to cope with complex situations, and that specific long and short-term goal setting improves performance. Fatigue in UER is multi-factorial, both physical and mental, and the perceived effort or level of fatigue have a major impact on the ability to continue at a given pace. Understanding the complex interplay of these limitations will help prepare UER competitors for the different scenarios they are likely to face. Therefore, this review takes an interdisciplinary approach to synthesising and illuminating limitations in UER performance to assist practitioners and scientists in making informed decisions in practice and applicable research.

Variation in human functional eccrine gland density and its implications for the evolution of human sweating.

OBJECTIVES: We aim to test three questions regarding human eccrine sweat gland density, which is highly derived yet poorly understood. First, is variation in functional eccrine gland density ("FED") explained by childhood climate, suggesting phenotypic plasticity? Second, is variation in FED explained by genetic similarity (a proxy for "geographic ancestry"), implying divergent evolutionary pathways in this trait of ancestral populations? Third, what is the relationship between FED and sweat production? MATERIALS AND METHODS: To test questions one and two, we measured FED in 68 volunteers aged 18-39 with varied childhood climate regimes and geographic ancestries. To test question three, we compared sweat production to FED in our n = 68 sample. In addition, we examined the relationship between FED and whole-body sweat loss during cycling in warm conditions using a sample of eight heat-acclimated endurance athletes. RESULTS: Interindividual variation in six-site FED was more than twofold, ranging from 60.9 to 132.7 glands/cm2 . Variation in FED was best explained by body surface area and limb circumferences (negative associations) and poorly explained by childhood climatic conditions and genetic similarity. Pilocarpine-induced sweat production was unrelated to FED while whole-body sweat loss during cycling was significantly, though modestly, associated with FED. DISCUSSION: We hypothesize that gland-level phenotypic plasticity, rather than changes in eccrine gland density, was sufficient to permit thermal adaptation to novel environments as humans colonized the globe. Future research should measure effects of FED in dehydrated states and the relationship between FED and salt loss, and control for effects of microclimate to rule out phenotypic plasticity effects.

How does Tra2ß protein regulate tissue-specific RNA splicing?

The splicing regulator protein Tra2ß is conserved between humans and insects and is essential for mouse development. Recent identification of physiological RNA targets has started to uncover molecular targets and mechanisms of action of Tra2ß. At a transcriptome-wide level, Tra2ß protein binds a matrix of AGAA-rich sequences mapping frequently to exons. Particular tissue-specific alternatively spliced exons contain high concentrations of high scoring Tra2ß-binding sites and bind Tra2ß strongly in vitro. These top exons were also activated for splicing inclusion in cellulo by co-expression of Tra2ß protein and were significantly down-regulated after genetic depletion of Tra2ß. Tra2ß itself seems to be fairly evenly expressed across several different mouse tissues. In the present paper, we review the properties of Tra2ß and its regulated target exons, and mechanisms through which this fairly evenly expressed alternative splicing regulator might drive tissue-specific splicing patterns.

Home Monitoring Programs for Patients Testing Positive for SARS-CoV-2: An Integrative Literature Review.

BACKGROUND: The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic threatened to oversaturate hospitals worldwide, necessitating rapid patient discharge to preserve capacity for the most severe cases. This need, as well as the high risk of SARS-CoV-2 transmission, led many hospitals to implement remote patient monitoring (RPM) programs for SARS-CoV-2 positive patients in an effort to provide care that was safe and preserve scarce resources. OBJECTIVE: The aim of this study is to provide an integrative review of peer-reviewed literature on different RPM programs that were implemented for SARS-CoV-2 positive patients including their strengths and challenges. METHODS: A search was conducted for peer reviewed literature using PubMed, CINAHL, OVID, and Google Scholar. Peer-reviewed studies written in English or Spanish and published between 2019 and 2021 on RPM of SARS-CoV-2-positive patients were considered. Information was extracted according to a qualitative content analysis method, informed by the Comparison of Mobile Patient Monitoring Systems Framework. RESULTS: Of 57 retrieved articles, 10 publications were included. The sample sizes ranged from 75 to 48,290 and the monitoring length ranged from 7 to 30 days. Information regarding the comparison framework was summarized. Main strengths of using RPM for SARS-CoV-2 positive patients was participant acceptance, feasibility, safety, and resource conservation. Main limitations were the lack of information on patient data security measures, robust outcomes testing, and identification of the most effective biomarkers to track SARS-CoV-2 decompensation. CONCLUSION: Different RPM programs for SARS-CoV-2 were implemented, from sending home participants with a pulse oximeter and collecting readings via call to modifying existing mobile applications and sending holistic health questionnaires to participants. This review determined that RPM is beneficial to SARS-CoV-2 positive patients; however, its effectiveness can be improved by further research. Mainly, identifying what patient data are most effective at tracking SARS-CoV-2 decompensation by utilizing advanced technology already in the market.

A comparison of proton stopping power measured with proton CT and x-ray CT in fresh postmortem porcine structures.

PURPOSE: Currently, calculations of proton range in proton therapy patients are based on a conversion of CT Hounsfield units of patient tissues into proton relative stopping power. Uncertainties in this conversion necessitate larger proximal and distal planned target volume margins. Proton CT can potentially reduce these uncertainties by directly measuring proton stopping power. We aim to demonstrate proton CT imaging with complex porcine samples, to analyze in detail three-dimensional regions of interest, and to compare proton stopping powers directly measured by proton CT to those determined from x-ray CT scans. METHODS: We have used a prototype proton imaging system with single proton tracking to acquire proton radiography and proton CT images of a sample of porcine pectoral girdle and ribs, and a pig's head. We also acquired close in time x-ray CT scans of the same samples and compared proton stopping power measurements from the two modalities. In the case of the pig's head, we obtained x-ray CT scans from two different scanners and compared results from high-dose and low-dose settings. RESULTS: Comparing our reconstructed proton CT images with images derived from x-ray CT scans, we find agreement within 1% to 2% for soft tissues and discrepancies of up to 6% for compact bone. We also observed large discrepancies, up to 40%, for cavitated regions with mixed content of air, soft tissue, and bone, such as sinus cavities or tympanic bullae. CONCLUSIONS: Our images and findings from a clinically realistic proton CT scanner demonstrate the potential for proton CT to be used for low-dose treatment planning with reduced margins.

Human chimeric antigen receptor macrophages for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.

Inferring User Intent using Bayesian Theory of Mind in Shared Avatar-Agent Virtual Environments.

We present a real-time algorithm to infer the intention of a user's avatar in a virtual environment shared with multiple human-like agents. Our algorithm applies the Bayesian Theory of Mind approach to make inferences about the avatar's hidden intentions based on the observed proxemics and gaze-based cues. Our approach accounts for the potential irrationality in human behavior, as well as the dynamic nature of an individual's intentions. The inferred intent is used to guide the response of the virtual agent and generate locomotion and gaze-based behaviors. Our overall approach allows the user to actively interact with tens of virtual agents from a first-person perspective in an immersive setting. We systematically evaluate our inference algorithm in controlled multi-agent simulation environments and highlight its ability to reliably and efficiently infer the hidden intent of a user's avatar even under noisy conditions. We quantitatively demonstrate the performance benefits of our approach in terms of reducing false inferences, as compared to a prior method. The results of our user evaluation show that 68.18% of participants reported feeling more comfortable in sharing the virtual environment with agents simulated with our algorithm as compared to a prior inference method, likely as a direct result of significantly fewer false inferences and more plausible responses from the virtual agents.