Design for Solubility May Reveal Induction of Amide Hydrogen/Deuterium Exchange by Protein Self-Association.

Structural heterogeneity often constrains the characterization of aggregating proteins to indirect or low-resolution methods, obscuring mechanistic details of association. Here, we report progress in understanding the aggregation of Adnectins, engineered binding proteins with an immunoglobulin-like fold. We rationally design Adnectin solubility and measure amide hydrogen/deuterium exchange (HDX) under conditions that permit transient protein self-association. Protein-protein binding commonly slows rates of HDX; in contrast, we find that Adnectin association may induce faster HDX for certain amides, particularly in the C-terminal ß-strand. In aggregation-prone proteins, we identify a pattern of very different rates of amide HDX for residues linked by reciprocal hydrogen bonds in the native structure. These results may be explained by local loss of native structure and formation of an inter-protein interface. Amide HDX induced by self-association, detected here by deliberate modulation of propensity for such interactions, may be a general phenomenon with the potential to expose mechanisms of aggregation by diverse proteins.

Emerging Evidence for Putative Neural Networks and Antecedents of Pediatric Anxiety in the Fetal, Neonatal, and Infant Periods.

Anxiety disorders are the most prevalent psychiatric disorders in youth and are associated with profound individual impairment and public health costs. Research shows that clinically significant anxiety symptoms manifest in preschool-aged children, and correlates of anxiety symptoms are observable in infancy. Yet, predicting who is at risk for developing anxiety remains an enduring challenge. Predictive biomarkers of anxiety are needed before school age when anxiety symptoms typically consolidate into diagnostic profiles. Increasing evidence indicates that early neural measures implicated in anxiety and anxious temperament may be incorporated with traditional measures of behavioral risk (i.e., behavioral inhibition) to provide more robust classification of pediatric anxiety problems. This review examines the phenomenology of anxiety disorders in early life, highlighting developmental research that interrogates the putative neurocircuitry of pediatric anxiety. First, we discuss enduring challenges in identifying and predicting risk for pediatric anxiety. Second, we summarize emerging evidence for putative neural antecedents and networks underlying risk for pediatric anxiety in the fetal, neonatal, and infant periods that represent novel potential avenues for risk identification and prediction. We focus on evidence examining the importance of early amygdala and extended amygdala circuitry development to the emergence of anxiety. Finally, we discuss the utility of integrating developmental psychopathology and neuroscience to facilitate future research and clinical work.

Phenoscreening: a developmental approach to research domain criteria-motivated sampling.

BACKGROUND: To advance early identification efforts, we must detect and characterize neurodevelopmental sequelae of risk among population-based samples early in development. However, variability across the typical-to-atypical continuum and heterogeneity within and across early emerging psychiatric/neurodevelopmental disorders represent fundamental challenges to overcome. Identifying multidimensionally determined profiles of risk, agnostic to DSM categories, via data-driven computational approaches represents an avenue to improve early identification of risk. METHODS: Factor mixture modeling (FMM) was used to identify subgroups and characterize phenotypic risk profiles, derived from multiple parent-report measures of typical and atypical behaviors common to autism spectrum disorder, in a community-based sample of 17- to 25-month-old toddlers (n = 1,570). To examine the utility of risk profile classification, a subsample of toddlers (n = 107) was assessed on a distal, independent outcome examining internalizing, externalizing, and dysregulation at approximately 30 months. RESULTS: FMM results identified five asymmetrically sized subgroups. The putative high- and moderate-risk groups comprised 6% of the sample. Follow-up analyses corroborated the utility of the risk profile classification; the high-, moderate-, and low-risk groups were differentially stratified (i.e., HR > moderate-risk > LR) on outcome measures and comparison of high- and low-risk groups revealed large effect sizes for internalizing (d = 0.83), externalizing (d = 1.39), and dysregulation (d = 1.19). CONCLUSIONS: This data-driven approach yielded five subgroups of toddlers, the utility of which was corroborated by later outcomes. Data-driven approaches, leveraging multiple developmentally appropriate dimensional RDoC constructs, hold promise for future efforts aimed toward early identification of at-risk-phenotypes for a variety of early emerging neurodevelopmental disorders.

Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol.

Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.

A Preliminary, Randomized-Controlled Trial of Mindfulness and Game-Based Executive Function Trainings to Promote Self-Regulation in Internationally-Adopted Children.

Although many children adopted internationally show remarkable recovery once placed in families, as a group they continue to exhibit persisting developmental deficits and delays in self-regulation. The current study uses a stratified, randomized, controlled trial to evaluate the effects of mindfulness-based and executive function trainings (EFTs) on internationally adopted (IA) children's self-regulation, including effortful/inhibitory control, attention, delay of gratification, and emotion-regulation. IA children ages 6-10 years were randomized into mindfulness training (MT), EFT, or no intervention (NI) groups. The MT and EFT groups attended 12 one-hour group sessions. Ninety-six children (MT, n = 33; EFT, n = 32; NI, n = 31) completed the study and were tested on computerized and non-computerized measures of self-regulation. Compared with the NI group, the MT group improved delay of gratification, and the EFT group improved inhibitory control and selective attention. There was no effect of either intervention on emotion regulation. MTs and EFTs show promise for improving self-regulation in IA children.

Persistent skewing of the T-cell profile in adolescents adopted internationally from institutional care.

The developing immune system is an adaptive system, primed by antigens, responsive to infectious pathogens, and can be affected by other aspects of the early rearing environment, including deviations from the normal provision of parental care. We investigated whether early rearing in an institutional setting, even when followed by years living in supportive and well-resourced families, would be associated with a persistent shift in T cell profiles. Immunophenotyping was used to enumerate CD4+ CD57+ and CD8+ CD57+ subsets, with gating strategies employed to differentiate naïve, central-memory, effector-memory, and terminally differentiated EM cells expressing CD45RA (TEMRA). Blood samples were collected from 96 adolescents, and PBMC isolated via Ficol gradient, followed by an optimized immunophenotypic characterization. CMV antibody titers were determined via ELISA. Adopted adolescents had lower CD4/CD8 ratios than did the control adolescents. Early rearing had a significant effect on the T cells, especially the CD8+ CD57+ CM, EM, and TEMRA cells and the CD4+ CD57+ EM cells. Adolescents who had spent their infancy in institutions before adoption were more likely to be seropositive for CMV, with higher antibody titers. CMV antibody titers were significantly correlated with the percentages of all CD8+ CD57+ cell subsets. In the statistical modeling, CMV antibody titer also completely mediated the relationship between institutional exposure and the ratio of CD4-to-CD8 cells, as well as the percentages of CD4+ CD57+ and CD8+ CD57+ subsets. These findings demonstrate that persistent immune differences are still evident even years after adoption by supportive American families. The shift in the T cells was associated with being a latent carrier of CMV and may reflect the role of specific T cell subsets in Herpes virus containment. In older adults, sustained CMV antigen persistence and immunoregulatory containment ultimately contributes to an accumulation of differentiated T cells with a decreased proliferative capacity and to immune senescence.

Social stress buffering by friends in childhood and adolescence: Effects on HPA and oxytocin activity.

Previous research has demonstrated that before puberty, parents are able to buffer, and often completely block, cortisol responses to social evaluative stressors (e.g., Trier Social Stress Test; TSST). However, after puberty, parents no longer provide a powerful buffer of the HPA axis from a social-evaluative stressor. The current study investigates whether friends can buffer the HPA axis in both children and adolescents compared to parents and whether similar stress-ameliorating patterns can also be observed in oxytocin activity. A total of 109 participants (54 children aged 9-10 and 55 adolescents aged 15-16; half of each sex) completed the TSST and were randomly assigned to prepare for their speech with their parent or friend for 5 minutes beforehand. Salivary cortisol and urinary oxytocin were measured before and after the TSST. For children, cortisol responses were comparable regardless of who helped the child prepare the speech. For adolescents, however, friends actually amplified the cortisol response compared to parents. In addition, adolescents produced less oxytocin than children, as did males compared to females. Notably, for boys, oxytocin levels decreased across the session if participants prepared with a friend rather than their parent. The mean change was in the same direction but not significant for girls. These results indicate that friends do not take over the social buffering role by age 15-16, which may inform interventions in at-risk children and adolescents.

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD12SH, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD12SH have shown that the ground state interconverts with a series of sparsely populated and transiently formed conformers, some of which have aberrant nonnative structures. Here, we study seven disease mutants of apoSOD12SH and characterize their free energy landscapes as a first step in understanding the initial stages of disease progression and, more generally, to evaluate the plasticity of low-lying protein conformational states. The mutations lead to little change in the structures and dynamics of the ground states of the mutant proteins. By contrast, the numbers of low-lying excited states that are accessible to each of the disease mutants can vary significantly, with additional conformers accessed in some cases. Our study suggests that the diversity of these structures can provide alternate interaction motifs for different mutants, establishing additional pathways for new and often aberrant intra- and intermolecular contacts. Further, it emphasizes the potential importance of conformationally excited states in directing both folding and misfolding processes.

The course of early disinhibited social engagement among post-institutionalized adopted children.

BACKGROUND: Approximately 20% of post-institutionalized (PI) children exhibit disinhibited social engagement (DSE) or the propensity to approach and engage strangers. There is little longitudinal research examining changes in DSE after adoption, or methods of identifying children with persistent behaviors. METHODS: DSE was assessed observationally four times during the first 2 years postadoption in PI children 16-36 months at adoption (n = 68) relative to same-age nonadopted children (n = 52). At age 5, a validated interview determined which PI children met criteria for Disinhibited Social Engagement Disorder (DSED). RESULTS: DSE trajectories initially increased and then stabilized. PIs had higher DSE levels initially and a steeper increase rate than NAs. When separated into physical and nonphysical DSE components, group differences arose in initial physical DSE and the rate of change of nonphysical DSE. DSE rate of increase predicted DSED diagnosis, as did longer institutional duration and poorer institutional care. CONCLUSIONS: The rate of increase in DSE postadoption, rather than the level observed at adoption, is predictive of disordered social engagement by age 5 years.

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer.

Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS). Despite being located throughout the protein, all mutations studied weaken the dimer interface, while concomitantly either decreasing or increasing the marginal stability of the monomer. Analysis of the populations of dimer, monomer, and unfolded monomer under physiological conditions of temperature, pH, and protein concentration shows that all mutations promote the formation of folded monomers. These findings may help rationalize the key roles proposed for monomer forms of SOD1 in neurotoxic aggregation in ALS, as well as roles for other forms of SOD1. Thus, the results obtained here provide a valuable approach for the quantitative analysis of homooligomeric protein stabilities, which can be used to elucidate the natural and aberrant roles of different forms of these proteins and to improve methods for predicting protein stabilities.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD1(2SH)). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD1(2SH) has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization.

Energetics of oligomeric protein folding and association.

In nature, proteins most often exist as complexes, with many of these consisting of identical subunits. Understanding of the energetics governing the folding and misfolding of such homooligomeric proteins is central to understanding their function and misfunction, in disease or biotechnology. Much progress has been made in defining the mechanisms and thermodynamics of homooligomeric protein folding. In this review, we outline models as well as calorimetric and spectroscopic methods for characterizing oligomer folding, and describe extensive results obtained for diverse proteins, ranging from dimers to octamers and higher order aggregates. To our knowledge, this area has not been reviewed comprehensively in years, and the collective progress is impressive. The results provide evolutionary insights into the development of subunit interfaces, mechanisms of oligomer folding, and contributions of oligomerization to protein stability, function and regulation. Thermodynamic analyses have also proven valuable for understanding protein misfolding and aggregation mechanisms, suggesting new therapeutic avenues. Successful recent designs of novel, functional proteins demonstrate increased understanding of oligomer folding. Further rigorous analyses using multiple experimental and computational approaches are still required, however, to achieve consistent and accurate prediction of oligomer folding energetics. Modeling the energetics remains challenging but is a promising avenue for future advances.

Regulation of the B cell receptor repertoire and self-reactivity by BAFF.

The TNF-family cytokine BAFF (BLyS) promotes B lymphocyte survival and is overexpressed in individuals with systemic lupus erythematosus and Sjögren's Syndrome. BAFF can rescue anergic autoreactive B cells from death, but only when competition from nonautoreactive B cells is lacking. Yet, high BAFF levels promote autoantibody formation in individuals possessing diverse B cells. To better understand how excess BAFF promotes autoimmunity in a polyclonal immune system, Ig L chain usage was analyzed in 3H9 site-directed IgH chain transgenic mice, whose B cells recognize DNA and chromatin when they express certain endogenous L chains. BAFF levels were manipulated in 3H9 mice by introducing transgenes expressing either BAFF or its natural inhibitor ΔBAFF. B cells in BAFF/3H9 mice were elevated in number, used a broad L chain repertoire, including L chains generating high-affinity autoreactivity, and produced abundant autoantibodies. Comparison of spleen and lymph node B cells suggested that highly autoreactive B cells were expanded. By contrast, ΔBAFF/3H9 mice had reduced B cell numbers with a repertoire similar to that of 3H9 mice, but lacking usage of a subset of Vκ genes. The results show that limiting BAFF signaling only slightly selects against higher affinity autoreactive B cells, whereas its overexpression leads to broad tolerance escape and positive selection of autoreactive cells. The results have positive implications for the clinical use of BAFF-depleting therapy.

The non-profit sector: leveraging resources and strengths to promote more physically active lifestyles.

BACKGROUND: Many Americans do not meet current minimum physical activity recommendations. Although the choice to be physically active is made by individuals, that choice is affected by the social and physical environments in which people live, work, play and learn. Creating environments that are more supportive of physical activity will require policies, practices and programs that individuals may not be able to influence on their own; such changes will require comprehensive, coordinated and collaborative action by a variety of organizational sectors at national, state and local levels. Because of their core-and frequently unique-competencies, many nonprofit organizations are poised to be active players in promoting important changes in policy and community environments that can facilitate lifelong physical activity for all Americans. METHODS: Review of mission statements and strategic plans of a variety of nonprofit organizations reveal key characteristics and competencies that can be leveraged, frequently across multiple levels and sectors, to promote physical activity. KEY RECOMMENDATIONS: Nonprofit organizations should leverage their unique capabilities, particularly in the areas of advocacy, strategic collaborations and outreach to their membership, volunteer and/or constituent bases to promote policy and environmental changes in support of physical activity.

The Non-Profit Sector: Leveraging Resources and Strengths to Promote More Physically Active Lifestyles.

BACKGROUND: Many Americans do not meet current minimum physical activity recommendations. Although the choice to be physically active is made by individuals, that choice is affected by the social and physical environments in which people live, work, play and learn. Creating environments that are more supportive of physical activity will require policies, practices and programs that individuals may not be able to influence on their own; such changes will require comprehensive, coordinated and collaborative action by a variety of organizational sectors at national, state and local levels. Because of their core-and frequently unique-competencies, many non-profit organizations are poised to be active players in promoting important changes in policy and community environments that can facilitate lifelong physical activity for all Americans. METHODS: Review of mission statements and strategic plans of a variety of non-profit organizations reveal key characteristics and competencies that can be leveraged, frequently across multiple levels and sectors, to promote physical activity. KEY RECOMMENDATIONS: Nonprofit organizations should leverage their unique capabilities, particularly in the areas of advocacy, strategic collaborations and outreach to their membership, volunteer and/or constituent bases to promote policy and environmental changes in support of physical activity.

Consequences of receptor editing at the lambda locus: multireactivity and light chain secretion.

To investigate the manner in which B cells with lambda light (L) chains undergo receptor editing, we have studied hybridoma panels from 56R/kappa-deleted (kdel) mice. 56R/kdel mice only produce four L chains (lambda1, lambda2, lambda3, and lambdaX). They also have a simplified heavy (H) chain repertoire: All B cells start out with a 56R anti-DNA H chain. A few frankly autoreactive 56R lambda1 cells appear to escape into the periphery, but the majority of the peripheral B cell repertoire in 56R/kdel is made up of B cells expressing the 56R H chain with the lambdaX L chain. Surprisingly, 56R lambdaX B cells are multireactive, binding to a variety of self and nonself antigens, including dsDNA (albeit at reduced affinity compared with the other lambda L chains). Another significant population in the 56R/kdel mouse consists of allelically included B cells that express lambdaX along with another L chain. The multireactivity of both 56R lambdaX and 56R lambdaX/lambda1 receptors could contribute to autoimmunity if these B cells were to become activated. Also found among 56R/kdel hybridomas are clones that have inactivated the H chain and secrete only L chains. These clones may represent products of exhaustive rearrangement. Multireactivity, allelic inclusion, and L chain secretion are three consequences of editing at the lambda locus that may predispose toward the development of autoimmunity.

Acetylation of cAMP-responsive element-binding protein (CREB) by CREB-binding protein enhances CREB-dependent transcription.

The coactivator function of cAMP-responsive element-binding protein (CREB)-binding protein (CBP) is partly caused by its histone acetyltransferase activity. However, it has become increasingly clear that CBP acetylates both histones and non-histone proteins, many of which are transcription factors. Here we investigate the role of CBP acetylase activity in CREB-mediated gene expression. We show that CREB is acetylated within the cell and that in vitro, CREB is acetylated by CBP, but not by another acetylase, p300/CBP-associated factor. The acetylation sites within CREB were mapped to three lysines within the CREB activation domain. Although inhibition of histone deacetylase activity results in an increase of CREB- or CBP-mediated gene expression, mutation of all three putative acetylation sites in the CREB activation domain markedly enhances the ability of CREB to activate a cAMP-responsive element-dependent reporter gene. Furthermore, these CREB lysine mutations do not increase interaction with the CRE or CBP. These data suggest that the transactivation potential of CREB may be modulated through acetylation by CBP. We propose that in addition to its functions as a bridging molecule and histone acetyltransferase, the ability of CBP to acetylate CREB may play a key role in modulating CREB-mediated gene expression.