Cdc2-like kinase 1 (CLK1) phosphorylates the serine-arginine (SR) proteins, a family of nuclear factors essential for mRNA splicing. The ability of CLK1 to recognize and efficiently modify SR proteins is strictly dependent on a lengthy, disordered N-terminus flanking its kinase domain. In addition to stimulating phosphorylation, this 150-residue extension also induces large oligomer formation in CLK1 but it is unclear whether said structure is important for catalytic or cellular function. We identified a subset of N-terminal residues that, upon removal, impairs oligomerization of CLK1 but does not abolish phosphorylation of the SR protein SRSF1. Despite robust phosphorylation, CLK1 lacking these high-order oligomerization sequences cannot effectively enter nuclear speckles and release SRSF1. This inability to mobilize CLK1 has detrimental effects on the alternative splicing of the CLK1 gene, severing an important, autoregulatory mechanism that controls active cellular levels of the kinase. Such findings indicate that whereas a limited group of residues in the N-terminus activates the kinase domain for SR protein phosphorylation, sequences that induce oligomerization direct CLK1 to the proper subnuclear structures for splicing function.

Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from Staphylococcus aureus, MTTSA (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 31 knot. Here, we report the non-proline backbone assignments with 98.8

Objective This study aimed to conceptualize teacher embodiment of mindfulness while teaching a mindfulness-based program at the elementary school level. It was predicated on the growing interest in such programs alongside the lack of easy-to-use standards and frameworks on mindfulness teacher competence in this setting. Method Building upon the Teacher Mindfulness in the Classroom framework, this paper presents a theory-building exploratory case study based on interviews of 11 elementary school teachers delivering Flourish : the Compassionate Schools Project curriculum. Results A conceptual framework, Mindfully Teaching Mindfulness—Elementary (MTM-E) is proposed and applied to two comparable classroom observations for illustration purposes. Conclusions This framework may be helpful for designing effective professional learning opportunities, promoting personal and professional practices among instructors, and informing the development of observational tools for assessing how well teachers embody mindfulness while teaching mindfulness in elementary school settings.

The prosocial classroom model emphasizes the importance of educators' social and emotional competencies (SEC) in relation to their capacity to cultivate and maintain socially and emotionally supportive environments and effectively promote students’ social and emotional learning (SEL). Beyond direct SEL instruction, educators must intentionally model SEL skills in their interactions with students while also promoting prosocial interactions among students. Furthermore, the social and emotional demands of the classroom may provoke high levels of occupational stress that may interfere with these learning processes. Given these demands, educators may need additional support to perform this role and limited research has explored the various facets of SEC required to do so. Leveraging insights regarding emotional schemas drawn from the metacognitive and meta-emotion literatures, this article seeks to address this gap. Identifying the construct of emotional schemas and its role in classroom interactions and applying it to educational contexts may lead to valuable new innovations to support educator SEC and student SEL. We describe emotional schemas and how they develop during socialization. We address dimensions of adaptability and variation across cultures. We discuss the potential impact of educators’ emotional schemas may have on stress and educators’ capacity to model and instruct SEL in the classroom context. We review evidence demonstrating the promise of several related intervention strategies that may support educators’ SEC and SEL instruction. We conclude by examining the potential impact the understanding of educators’ emotional schemas may have on both practice and policy, and by proposing recommendations for future research.

Pulmonary arterial hypertension (PAH) is associated with significant morbidity and mortality. PAH is characterized by pulmonary artery remodeling, elevated right ventricular pressure (RVP) and, ultimately, cardiac failure. Pulmonary endothelial cells can sense danger or damage caused by mechanical injury or pathogens through alarmin cytokines. These cytokines can signal proliferation to restore barrier integrity or aberrant hyperproliferation and remodeling. We hypothesized that IL-33 signals pulmonary artery endothelial cells to proliferate under hypertensive conditions during the remodeling response and rise in RVP. To test this hypothesis, pulmonary hypertension (PH) was induced in C57Bl/6J, IL-33 receptor gene deleted (ST2-/- ) and MYD88 gene deleted (MYD88-/- ) mice by exposure to 10% O2 and SU5416 injections (SUHX). RVP, arterial wall thickness, endothelial cell proliferation and IL-33 levels and signaling were evaluated. In response to SUHX. RVP increased in C57Bl/6J mice in response to SUHX (49% male and 70% female; p < 0.0001) and this SUHX response was attenuated in ST2-/- mice (29% male p = 0.003; 30% female p = 0.001) and absent in MYD88-/- mice. Wall thickness was increased in SUHX C57Bl/6J mice (p = 0.005), but not in ST2-/- or MYD88-/- mice. Proliferating cells were detected in C57Bl/6J mice by flow cytometry (CD31+ /BrDU+ ; p = 0.02) and immunofluorescence methods (Ki-67+). IL-33 was increased by SUHX (p = 0.03) but a genotype effect was not observed (p = 0.76). We observed that in hPAECs, IL-33 expression is regulated by both IL-33 and DLL4. These data suggest IL-33/ST2 signaling is essential for the endothelial cell proliferative response in PH.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.

Conformational changes are an essential feature for the function of some dynamic proteins. Understanding the mechanism of such motions may allow us to identify important properties, which may be directly related to the regulatory function of a protein. Also, this knowledge may be employed for a rational design of drugs that can shift the balance between active and inactive conformations, as well as affect the kinetics of the activation process. Here, the conformational changes in carboxyl-terminal Src kinase, the major catalytic repressor to the Src family of kinases, was investigated, and it was proposed as a functionally related hypothesis. A Cα Structure-Based Model (Cα-SBM) was applied to provide a description of the overall conformational landscape and further analysis complemented by detailed molecular dynamics simulations. As a first approach to Cα-SBM simulations, reversible transitions between active (closed) and inactive (open) forms were modeled as fluctuations between these two energetic basins. It was found that, in addition to the interdomain Carboxyl-terminal SRC Kinase (Csk) correlated motions, a conformational change in the αC helix is required for a complete conformational transition. The result reveals this as an important region of transition control and domain coordination. Restrictions in the αC helix region of the Csk protein were performed, and the analyses showed a direct correlation with the global conformational changes, with this location being propitious for future studies of ligands. Also, the Src Homology 3 (SH3) and SH3 plus Src Homology 2 (SH2) domains were excluded for a direct comparison with experimental results previously published. Simulations where the SH3 was deleted presented a reduction of the transitions during the simulations, while the SH3-SH2 deletion vanishes the Csk transitions, corroborating the experimental results mentioned and linking the conformational changes with the catalytic functionality of Csk. The study was complemented by the introduction of a known kinase inhibitor close to the Csk αC helix region where its consequences for the kinetic behavior and domain displacement of Csk were verified through detailed molecular dynamics. The findings describe the mechanisms involving the Csk αC helix for the transitions and also support the dynamic correlation between SH3 and SH2 domains against the Csk lobes and how local energetic restrictions or interactions in the Csk αC helix can play an important role for long-range motions. The results also allow speculation if the Csk activity is restricted to one specific conformation or a consequence of a state transition, this point being a target for future studies. However, the αC helix is revealed as a potential region for rational drug design.

To date, there has been a lag between the rise in E-cigarette use and an understanding of the long-term health effects. Inhalation of E-cigarette aerosol delivers high doses of nicotine, raises systemic cytokine levels, and compromises cardiopulmonary function. The consequences for muscle function have not been thoroughly investigated. The present study tests the hypothesis that exposure to nicotine-containing aerosol impairs locomotor muscle function, limits exercise tolerance, and interferes with muscle repair in male mice. Nicotine-containing aerosol reduced the maximal force produced by the extensor digitorum longus (EDL) by 30%-40% and, the speed achieved in treadmill running by 8%. Nicotine aerosol exposure also decreased adrenal and increased plasma epinephrine and norepinephrine levels, and these changes in catecholamines manifested as increased muscle and liver glycogen stores. In nicotine aerosol exposed mice, muscle regenerating from overuse injury only recovered force to 80% of noninjured levels. However, the structure of neuromuscular junctions (NMJs) was not affected by e-cigarette aerosols. Interestingly, the vehicle used to dissolve nicotine in these vaping devices, polyethylene glycol (PG) and vegetable glycerin (VG), decreased running speed by 11% and prevented full recovery from a lengthening contraction protocol (LCP) injury. In both types of aerosol exposures, cardiac left ventricular systolic function was preserved, but left ventricular myocardial relaxation was altered. These data suggest that E-cigarette use may have a negative impact on muscle force and regeneration due to compromised glucose metabolism and contractile function in male mice.NEW & NOTEWORTHY In male mice, nicotine-containing E-cigarette aerosol compromises muscle contractile function, regeneration from injury, and whole body running speeds. The vehicle used to deliver nicotine, propylene glycol, and vegetable glycerin, also reduces running speed and impairs the restoration of muscle function in injured muscle. However, the predominant effects of nicotine in this inhaled aerosol are evident in altered catecholamine levels, increased glycogen content, decreased running capacity, and impaired recovery of force following an overuse injury.

The quality of students' relationships with their teachers plays a significant role in their success in school. Social and emotional learning (SEL) curriculums show great promise for supporting student development. However, quality implementation requires that teachers recognize and understand how their behavior and interactions with students impact the development of these skills. The Prosocial Classroom Model proposes that teacher social and emotional competencies (SECs) play a critical role in creating and maintaining a classroom where everyone feels safe, connected, and engaged in learning. In this chapter, we extend the understanding of SEC to include leadership styles as defined by evolutionary motivational systems theory. We argue that a critical dimension of effective SEL instruction and teacher SEC is effective leadership that skillfully applies an understanding of the social and emotional dimensions of classroom interactions that promote motivation, engagement, and learning. Implications for educational theory, policy and practice, and research are discussed.

Early care and education (ECE) professionals' social and emotional well-being has emerged as an area of focus for improving program quality. ECE professionals are experiencing increasing levels of stress and burnout, which is threatening the quality of early childhood education and care. This article introduces the special issue devoted to understanding early childhood professionals' social and emotional competence and well-being and their relationship to program quality and child outcomes. The purposes of this special issue are to (a) describe the nature of ECE professionals' social and emotional competence and well-being, (b) understand the factors that contribute to ECE professionals' social and emotional competence and well-being, (c) understand to what extent and how ECE professionals' social and emotional competence and well-being impacts classroom quality as well as children's developmental outcomes, and (d) present evidence-based approaches that support ECE professionals' social and emotional competence and well-being. For the purposes of this issue, ECE professionals include early childhood educators (e.g., teachers or home-based child care providers), practitioners (e.g., home visitors, mental health consultants, specialists, etc.) and early childhood leaders (e.g., directors, principals, or administrators) who serve children from birth to third grade. In this introductory article we conclude with a discussion of implications of this research for future research, policy, and practice.

Research Findings Developing students' social and emotional skills positively affects their academic success. The Prosocial Classroom model (PCM) highlights the importance of teacher's social-emotional competency (SEC) and well-being. The current research extends the PCM by using the Five Awarenesses of Teaching Framework (The Framework) to understand teacher SEC through a developmental lens. We discuss how the Framework can inform and be informed by our understanding of Early Care and Education (ECE) teachers' SEC. Eighteen ECE teachers participated in cognitive interviews and a deductive thematic analysis was used to categorize teachers' interview data according to The Framework. Three key themes were identified: 1) The Framework described cognitive capacities relevant to ECE teacher SEC consistent with other teacher populations; 2) there was an underlying conflict between teachers' keen awareness of student social-emotional learning (SEL) and an active suppression of their own social and emotional well-being; and 3) the impact and importance of race, ethnicity, and family engagement.Practice or Policy: Findings reveal that ECE teachers' SEC and well-being can be described using The Framework. Additionally, the suppression of ECE teacher emotional needs and the importance of family engagement suggest important avenues for improving our understanding of ECE SEC and well-being.

Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.

The methyltransferases that belong to the SpoU-TrmD family contain trefoil knots in their backbone fold. Recent structural dynamic and binding analyses of both free and bound homologs indicate that the knot within the polypeptide backbone plays a significant role in the biological activity of the molecule. The knot loops form the S-adenosyl-methionine (SAM)-binding pocket as well as participate in SAM binding and catalysis. Knots contain both at once a stable core as well as moving parts that modulate long-range motions. Here, we sought to understand allosteric effects modulated by the knotted topology. Uncovering the residues that contribute to these changes and the functional aspects of these protein motions are essential to understanding the interplay between the knot, activation of the methyltransferase, and the implications in RNA interactions. The question we sought to address is as follows: How does the knot, which constricts the backbone as well as forms the SAM-binding pocket with its three distinctive loops, affect the binding mechanism? Using a minimally tied trefoil protein as the framework for understanding the structure-function roles, we offer an unprecedented view of the conformational mechanics of the knot and its relationship to the activation of the ligand molecule. Focusing on the biophysical characterization of the knot region by NMR spectroscopy, we identify the SAM-binding region and observe changes in the dynamics of the loops that form the knot. Importantly, we also observe long-range allosteric changes in flanking helices consistent with winding/unwinding in helical propensity as the knot tightens to secure the SAM cofactor.

Pulmonary arterial hypertension (PAH) is a devastating disease that is often misdiagnosed and has a high mortality rate. It is characterized by remodeling of the small resistance arteries in the lungs that leads to elevated right ventricular pressure (RVP) and cardiac failure. Interleukin‐33 (IL‐33) is a cytokine that functions as an alarmin to sense danger or damage to endothelial cells, signal proliferation and restore barrier integrity. However, uncontrolled, active forms of IL‐33 could initiate endothelial hyperproliferation, a putative first step in the remodeling process. We hypothesize that IL‐33 initiates the hyperproliferative response of pulmonary artery endothelial cells and contributes to the wall remodeling and increased RVP observed in PAH. To test this, we induced pulmonary hypertension of C57Bl/6J (wild‐type), IL‐33 receptor knock‐out (ST2KO) and the adaptor protein, MyD88 knock‐out (MyD88KO) mice by exposure to 10% O2 and weekly SU5416 injections (SuHx). Hypertension was assessed by measuring RVP and arterial wall thickness (ratio of wall cross‐sectional area/total vessel cross‐sectional area). Proliferating pulmonary endothelial (CD31+/BrdU+) cells were detected by flow cytometry. Processed IL‐33 forms in the lung were analyzed by western blot. RVPs increased in wild‐type mice in response to SuHx (C57Bl/6J male, RA/DMSO 27.0 ± 3.0 mmHg, SuHx 40.5 ± 6.3 mmHg, p < 0.01; female, RA/DMSO 25.8 ± 3.3 mmHg, SuHx 43.8 ± 7.4 mmHg, p < 0.01) and, this response was attenuated in ST2KO mice (ST2KO male, RA/DMSO 25.0 ± 4.3 mmHg, SuHx 32.8 ± 6.9 mmHg, p<0.01; female, RA/DMSO 25.3 ± 3.5 mmHg, SuHx 32.8 ± 4.9 mmHg, p<0.01) and completely blocked in MyD88KO mice (MyD88KO male, RA/DMSO 19 ± 2.9 mmHg, SuHx 19.7 ± 5.9 mmHg, NS; female, RA/DMSO 20.2 ± 2.5 mmHg, SuHx 24.5 ± 7.1 mmHg, NS). Similarly, wall thickness increased in wild‐type mice, but not ST2KO and MyD88KO mice, under SuHx conditions (C57Bl/6J Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, p < 0.01; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, p=0.4; ST2KO Male, RA/DMSO 0.37 ± 0.07, SuHx 0.38 ± 0.13, NS; Female RA/DMSO 0.31 ± 0.02, SuHx 0.34 ± 0.03, NS; MyD88KO Male, RA/DMSO 0.37 ± 0.05, SuHx 0.41 ± 0.10, NS; Female RA/DMSO 0.34 ± 0.07, SuHx 0.38 ± 0.03, NS). The percentage of proliferating endothelial cells was increased in wild‐type mice under SuHx conditions (C57Bl/6J Male, RA/DMSO 1.9 ± 0.4, SuHx 4.8 ± 2.6, p< 0.01; Female RA/DMSO 0.3 ± 0.05, SuHx 0.4 ± 0.1, p= 0.07). This endothelial proliferative response was absent in ST2KO and Myd88KO mice (ST2KO Male, RA/DMSO 0.35 ± 0.07, SuHx 0.53 ± 0.12, NS; Female RA/DMSO 0.34 ± 0.05, SuHx 0.41 ± 0.1, NS). Proliferating (Ki‐67+) cells were detected in the intima layer of small arteries. The C‐terminal and caspase cleaved form of IL‐33 is increased in wild‐type and ST2KO mice but not in MyD88KO mice under SuHx conditions. IL‐33 signaling through the ST2 receptor is important for endothelial cell proliferation in the SuHx model. This response is associated with increased levels of both the C‐terminal IL‐1 like cytokine domain and caspase cleaved fragment of IL‐33 in lung tissue.Support or Funding InformationDepartment of Veteran Affairs

Chronic obstructive pulmonary disease (COPD) is a worldwide threat. Cigarette smoke (CS) exposure causes cardiopulmonary disease and COPD and increases the risk for pulmonary tumors. In addition to poor lung function, patients with COPD are susceptible to bouts of dangerous inflammation triggered by pollutants or infection. These severe inflammatory episodes can lead to additional exacerbations, hospitalization, further deterioration of lung function, and reduced survival. Suitable models of the inflammatory conditions associated with CS, which potentiate the downward spiral in patients with COPD, are lacking, and the underlying mechanisms that trigger exacerbations are not well understood. Although initial CS exposure activates a protective role for vascular endothelial growth factor (VEGF) functions in barrier integrity, chronic exposure depletes the pulmonary VEGF guard function in severe COPD. Thus, we hypothesized that mice with compromised VEGF production and challenged with CS would trigger human-like severe inflammatory progression of COPD. In this model, we discovered that CS exposure promotes an amplified IL-33 cytokine response and severe disease progression. Our VEGF-knockout model combined with CS recapitulates severe COPD with an influx of IL-33-expressing macrophages and neutrophils. Normally, IL-33 is quickly inactivated by a post-translational disulfide bond formation. Our results reveal that BAL fluid from the CS-exposed, VEGF-deficient cohort promotes a significantly prolonged lifetime of active proinflammatory IL-33. Taken together, our data demonstrate that with the loss of a VEGF-mediated protective barrier, the CS response switches from a localized danger to an uncontrolled long-term and long-range, amplified, IL-33-mediated inflammatory response that ultimately destroys lung function.

The articles in this special issue demonstrate that early childhood educator psychological distress is a growing problem and shed light on the causes of this distress. Further, they illuminate the negative impacts early childhood educator distress can have on young children. However, there is still much to learn about how best to support teachers in order to reduce psychological distress, promote social and emotional skills and promote positive impacts on young children. Addressing these complex challenges will require long-term systemic change and the urgent nature of the problem will require strategic prioritization to address the immediate needs as well as long-term goals. In this concluding article, promising approaches designed to address the problem at the individual- and systems-level are presented.

This study examined the cost of implementing the Cultivating Awareness and Resilience in Education (CARE) for Teachers professional development program during a randomized controlled trial targeting a diverse sample of public elementary school teachers in New York City. Detailed budget information collected during the study was used to identify the cost of all necessary resources associated with program implementation. The largest expense category was opportunity costs associated with teacher participation, accounting for over 40% of the total cost. This was closely followed by the program-required costs related to coordination, facilitation, and supplies for the program (31.8%). Finally, ancillary costs related to facilitator travel, room rental, and food for the program implementations encompassed 11% of the total cost. Across all three program implementations, 118 teachers were trained; the average cost per teacher was US$1217 when accounting for all categories. In future cost projections for a training with 30 teachers, the price per teacher is only US$515 when considering program-required and indirect costs. The costs for implementing the CARE for Teachers program are similar to those reported for other mindfulness-based interventions (MBIs). This paper provides a detailed analysis of the full cost of providing an evidence-based MBI to teachers in a public education setting. This research can help inform communities interested in funding future CARE for Teachers program implementations, provide an example for cost reporting for other MBIs, and provide a basis for future cost-effectiveness and benefit-cost analyses.