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This qualitative study explores the aspects of a residential wilderness experience that informed self-evaluations in male adolescents, ages 12-16. To assess change in self-evaluations and program factors associated with change, qualitative interviews were conducted with adolescents upon entry to the program and four months later. Participants' responses suggested that self-evaluation improved in relation to specific factors in the residential wilderness program. Specifically, increased levels of social support provided through cooperative experiences and opportunities for emotional expression helped participants develop a more positive sense of self. Findings contribute to our knowledge of specific program components offered by residential wilderness programs that are associated with positive adjustment in adolescents. [ABSTRACT FROM AUTHOR]

The authors investigate whether parental use of punitive discipline and yielding to coercion varies in levels and associated child outcomes for mothers with different parenting styles. Participants were fourth-grade children (N = 370) and their mothers. Maternal parenting style was determined based on levels of responsiveness and demandingness. Authoritative mothers used less punitive discipline than indifferent mothers. Authoritative and authoritarian mothers engaged in less yielding to coercion than indifferent or indulgent mothers. More punitive discipline and yielding to coercion were associated with lower academic grades and more punitive discipline was associated with more social problems, with these effects not moderated by parenting style. Negative effects of yielding to coercion in terms of internalizing, externalizing, and social problems were observed only within authoritarian families. Greater use of punitive discipline was associated with more externalizing problems within the indulgent and authoritarian parenting style groups and more internalizing problems within the authoritarian group. [ABSTRACT FROM AUTHOR]

Intimate partner violence (IPV) is common, and almost half of all IPV takes place in relationships with children in the home. We inventoried laws in the 50 states and the District of Columbia in the United States of America (USA) focused on addressing IPV committed in the presence of children, as these laws could help prevent or remediate this critical health and social issue. Using WestLaw, a web-based legal research service, we identified over 1,200 statutes and 500 regulations. We documented the laws' key attributes and heterogeneities and coded 557 laws from 31 states. We determined that the most commonly prescribed penalty was stricter sentencing, followed by mandates to pay for counseling for any child witnesses, separate additional criminal charges, mandated receipt of counseling or intervention services, and a period of supervised parenting. Future research could assess the possible impacts of these laws on children's short- and long-term wellbeing., Competing Interests: Declarations. Conflict of interest: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objective: Covid-19 has resulted in changes to college students' daily lives due to recommendations to socially distance. Social distance is likely to affect youths' peer relationships at a time when these relationships remain important for development. Participants and Methods: The current study utilized survey data to examine perceived changes in peer relationships and the association with behavioral health among 275 college students ( Mage  = 18.69, SD =.72) during the fall/spring of 2020-2021. Results: Quantitative results indicated that participants reported significant perceived decreases in peer support but did not report significantly worse quality of closest friendships. Qualitative responses from an open-ended survey question supported these quantitative results suggesting that although many participants perceived decreases in quality of relationships, there was also the perception that friendships became closer. Participants who perceived decreased peer relationship support and quality were more likely to report depressive symptoms and loneliness when compared to participants who perceived no changes.

Positive identification and reporting of therapeutic substances intended for human consumption in race-day equine and canine samples is a controversial topic. While inadvertent environmental exposure is a potential cause for the presence of these substances in race-day samples, intentional use cannot be ruled out given their therapeutic benefits. Pregabalin is widely prescribed in Australia to treat epilepsy, anxiety, and neuropathic pain in humans; however, it is also increasingly used as a recreational drug. Metformin is commonly used to treat type 2 diabetes in humans. Both pregabalin and metformin have no routine use on racing animals and should not be present in race-day samples taken from healthy animals. They are prohibited substances under the Rules of Racing with no established screening limits. Although therapeutic levels for these substances have been established in humans, such information is not available for animals. Pregabalin and metformin are analytically challenging molecules, more so when they are extracted from biological matrices routinely screened for hundreds of other compounds simultaneously. A simple extraction, followed by a targeted Ultra High-Pressure Liquid Chromatography Orbitrap™ Mass Spectrometry method utilising a reverse-phase C18 column, is presented. This method is effective in screening for pregabalin and metformin, in addition to more than 150 other compounds of interest in equine and canine urines. The prevalence of pregabalin and metformin in race-day equine and canine urine samples in Western Australia was monitored using this method over 12 months. More than 4000 urine samples were screened, and four samples were confirmed positive for these substances., (© 2024 John Wiley & Sons Ltd.)

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions., (© 2020 Wiley Periodicals LLC.)

We present a method to stabilize two lasers to an optical cavity using pulsed triple frequency modulation. The setup allows simultaneous Pound-Drever-Hall stabilization, as well as independent frequency control, while removing interference terms that limit the frequency scan range and allowing for smaller modulation depths. A review of single, dual, and triple frequency modulation is also presented in addition to a discussion of how to effectively turn pulsed triple frequency modulation into independent dual frequency modulation for each laser. This method would increase the scan range to half the free spectral range.

The inability to spatiotemporally guide proteins in tissues and efficiently deliver them into cells remains a key barrier to realizing their full potential in precision medicine. Here, we report ultrasound-sensitive fluoro-protein nanoemulsions which can be acoustically tracked, guided, and activated for on-demand cytosolic delivery of proteins, including antibodies, using clinically relevant diagnostic ultrasound. This advance is accessed through the discovery of a family of fluorous tags, or FTags, that transiently mask proteins to mediate their efficient dispersion into ultrasound-sensitive liquid perfluorocarbons, a phenomenon akin to dissolving an egg in liquid Teflon. We identify the biochemical basis for protein fluorous masking and confirm FTag coatings are shed during delivery, without disrupting the protein structure or function. Harnessing the ultrasound sensitivity of fluorous emulsions, real-time imaging is used to simultaneously monitor and activate FTag-protein complexes to enable controlled cytosolic antibody delivery in vitro and in vivo . These findings may advance the development of image-guided, protein-based biosensing and therapeutic modalities.

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum , Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4 + and CD8 + T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

Laboratory models that help us understand the neural mechanisms associated with how stress, particularly interpersonal stress, affects children's and adolescents' emotions are paramount but are limited if that understanding lacks validity in adolescents' daily lives. There is a lack of research that addresses the ecological validity of visual stimuli to induce stress in participants while measuring participants' neural response to that stimuli. This approach is needed if we are to identify the neural mechanisms that underlie the effect of stressful events on individuals' emotional functioning. The current study conducted a systematic literature review to identify visual tasks that have been used in laboratory settings to induce stress in participants. The most frequent tasks identified were developed to induce peer rejection/exclusion in youth (e.g., Chatroom and Cyberball). These tasks were generally effective at bringing about a neural response in areas of the brain traditionally associated with social cognitive processing, such as the cingulate cortex, prefrontal cortex, insula, and striatum. In particular, the cingulate cortex and prefrontal cortex are associated with the Social Information Processing Network. Almost entirely absent from the literature are systematic evaluations of ecological validity and parent-child based visual stimuli that approximate the stress that adolescents might experience in their relationships with parents. The present article highlights trends and gaps in the current research, and examines the ecological validity of current stimuli used as laboratory based stressors, which can be used to fuel further investigation into adolescent neural response to stimuli, and further evaluation of the ecological validity of tasks., Competing Interests: Conflict of interest The authors report no conflicts of interest.

Objective: To undertake the first detailed genomic analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Sri Lanka. Methods: A prospective observational study was performed on 94 MRSA isolates collected over a 4 months period from the Anuradhapura Teaching Hospital, Sri Lanka. Screening for mec A, mec C, and the Panton-Valentine leucocidin (PVL)-associated lukS-PV/lukF-PV genes and molecular characterization by spa typing was undertaken. Whole genome sequencing (WGS) and phylogenetic analysis was performed on selected multilocus sequence type (MLST) clonal complex 5 (CC5) isolates from Sri Lanka, England, Australia, and Argentina. Results: All 94 MRSA harbored the mecA gene. Nineteen spa types belonging to nine MLST clonal complexes were identified. Where origin of the sample was recorded, most isolates were from skin and soft tissue infections (70/91; 76.9%), with fewer causing bacteremia (16/91; 17.6%), empyema (3/91; 3.3%) and osteomyelitis (2/91; 2.2%). Sixty two (65.9%) isolates were PVL positive with the majority (56 isolates; 90.3%) belonging to a dominant CC5 lineage. This lineage, PVL-positive ST5-MRSA-IVc, was associated with both community and hospital-onset infections. Based on WGS, representative PVL-positive ST5-MRSA-IVc isolates from Sri Lanka, England and Australia formed a single phylogenetic clade, suggesting wide geographical circulation. Conclusions: We present the most detailed genomic analysis of MRSA isolated in Sri Lanka to date. The analysis identified a PVL-positive ST5-MRSA-IVc that is prevalent among MRSA causing clinical infections in Sri Lanka. Furthermore, this clone was also found among isolates from the United Kingdom and Australia.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with Ca II binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for Ln III s and Y III over Ca II . Here we present the nuclear magnetic resonance solution structure of LanM complexed with Y III . This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for Ln III s, and it suggests a role of unusual N i+1 -H···N i hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective Ln III recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.

The pancreatic and duodenal homeobox 1 (Pdx1) is an essential pancreatic transcription factor. The C-terminal intrinsically disordered domain of Pdx1 (Pdx1-C) has a heavily biased amino acid composition; most notably, 18 of 83 residues are proline, including a hexaproline cluster near the middle of the chain. For these reasons, Pdx1-C is an attractive target for structure characterization, given the availability of suitable methods. To determine the solution ensembles of disordered proteins, we have developed a suite of 13 C direct-detect NMR experiments that provide high spectral quality, even in the presence of strong proline enrichment. Here, we have extended our suite of NMR experiments to include four new pulse programs designed to record backbone residual dipolar couplings in a 13 C, 15 N-CON detection format. Using our NMR strategy, in combination with small-angle X-ray scattering measurements and Monte Carlo simulations, we have determined that Pdx1-C is extended in solution, with a radius of gyration and internal scaling similar to that of an excluded volume polymer, and a subtle tendency toward a collapsed structure to the N-terminal side of the hexaproline sequence. This structure leaves Pdx1-C exposed for interactions with trans-regulatory co-factors that contribute with Pdx1 to transcription control in the cell.

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The calcineurin (CaN) regulatory domain (RD) is responsible for regulating the enzyme's phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorder-to-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating calmodulin (CaM) binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder facilitate CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We report spectroscopic results on the 2s2p^{1}P_{1} state in neutral atomic beryllium-9. The absolute frequency for the center of gravity is determined to be 42 565.4501(13)  cm^{-1}, a factor of 130 more precise than the previous experimental measurement. The result is in agreement with and a factor of 8 more precise than the current best theoretical estimate of 42 565.441(11)  cm^{-1}, which was calculated including the effects of quantum electrodynamics. Because of the large natural linewidth of the transition, the hyperfine constants were not able to be extracted to high precision.

Few laboratory paradigms exist that expose adolescents to conflict that might commonly be experienced in parent-adolescent relationships. Given the continued importance of parent-adolescent relationships on adolescent development, as well as the changing expectations in these relationships, we examined the effect of a novel parent-adolescent conflict paradigm on physiological and affective response in a sample of 52 adolescents. The parent-adolescent conflict stressor (PACS) involved adolescent participants (50% girls; M = 14.75, SD = 0.88) watching a 12-minute scripted video that asked youth to imagine that they were the teenager in the video, which consisted of parent and adolescent actors having discussions about conflict in their relationship and solving this conflict in either a positive, typical, or hostile manner. Cortisol, alpha amylase, and self-report of negative and positive affect were collected at baseline, following the video, and during a recovery period. Heart rate also was taken continuously while adolescents watched the videos. Hierarchical linear modeling (HLM) analyses indicated significant linear change in alpha amylase and linear and quadratic change in negative affect to the PACS. There also was a significant linear and quadratic change in heart rate during the portion of the video where teens and parents discussed issues of personal responsibility. The PACS marks a preliminary but important first step in developing a parent-adolescent conflict paradigm that can be used across studies to understand the impact of parent-adolescent conflict on affective and physiological markers associated with stress response.

Developing autonomy is an important developmental task that has implications for adolescent adjustment and may be impacted by adolescents' response to stress. This study examined whether stress reactivity (i.e., cortisol and heart rate reactivity) to a parent-adolescent conflict interaction moderates the effect of autonomy on adjustment assessed one year later in 100 adolescents (M age = 15.09; 68% girls). Multiple group models suggested that youth who evidenced higher stress reactivity when compared to those with lower stress reactivity were more likely to report decreased externalizing problems and internalizing problems when their parents granted more autonomy. In contrast, youth who evidenced higher stress reactivity who experienced undermining of autonomy were more likely to report increased externalizing and internalizing problems than youth who evidenced lower stress reactivity. Results support biological sensitivity to context theory and highlight the importance of considering individual differences in the effect of developmental milestones on adolescents' adjustment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Understanding individual differences in adolescents' ability to regulate emotions within interpersonal relationships is paramount for healthy development. Thus, the effect of individual vulnerabilities (depressive affect, social anxiety, self-blame, and coping efficacy problems) on the transmission of emotional reactivity in response to conflict from family to peers (friends and romantic partners) was prospectively examined across six waves of data in a community-based sample of 416 adolescents (M age Wave 1 = 11.90, 51% girls). Multiple-group models estimated in structural equation modeling suggested that youth who were higher in social anxiety or coping efficacy problems were more likely to transmit emotional reactivity developed in the family-of-origin to emotional reactivity in response to conflict in close friendships. Additionally, those youth higher in self-blame and depressive affect were more likely to transmit emotional reactivity from friendships to romantic relationships.

NMR spectroscopy remains the only experimental technique that provides (near) atomistic structural information for intrinsically disordered proteins (IDPs), but their sequence and structure characteristics still pose major challenges for high-resolution spectroscopy. Carbon-13 direct-detect NMR spectroscopy can overcome poor spectral dispersion and other difficulties associated with traditional 1 H-detected NMR of nonaggregating disordered proteins. This chapter presents spectroscopic protocols suitable for complete characterization of IDPs that rely exclusively on 13 C direct-detect experiments. The protocols described span initial characterization and chemical shift assignment; structure constraint through residual dipolar coupling and paramagnetic relaxation enhancement measurements; and assessment of intramolecular dynamics through 15 N spin relaxation. The experiments described empower investigators to establish molecular mechanisms and structure-function relationships for IDPs and other proteins characterized by high internal flexibility., (© 2018 Elsevier Inc. All rights reserved.)

The positive transcription elongation factor b (P-TEFb) promotes transcription elongation through phosphorylation of the RNA polymerase II C-terminal domain. This process is not well understood, partly due to difficulties in determining the specificity of P-TEFb toward the various heptad repeat motifs within the C-terminal domain. A simple assay using mass spectrometry was developed to identify the substrate specificity of the Drosophila melanogaster P-TEFb (DmP-TEFb) in vitro. This assay demonstrated that DmP-TEFb preferentially phosphorylates Ser5 and, surprisingly, that pre-phosphorylation or conserved amino acid variation at the 7-position in the heptad can alter DmP-TEFb specificity, leading to the creation of distinct double-phosphorylation marks., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Calcineurin (CaN) plays an important role in T-cell activation, cardiac system development and nervous system function. Previous studies have demonstrated that the regulatory domain (RD) of CaN binds calmodulin (CaM) towards the N-terminal end. Calcium-loaded CaM activates the serine/threonine phosphatase activity of CaN by binding to the RD, although the mechanistic details of this interaction remain unclear. It is thought that CaM binding at the RD displaces the auto-inhibitory domain (AID) from the active site of CaN, activating phosphatase activity. In the absence of calcium-loaded CaM, the RD is disordered, and binding of CaM induces folding in the RD. In order to provide mechanistic detail about the CaM-CaN interaction, we have undertaken an NMR study of the RD of CaN. Complete 13 C, 15 N and 1 H assignments of the RD of CaN were obtained using solution NMR spectroscopy. The backbone of RD has been assigned using a combination of 13 C-detected CON-IPAP experiments as well as traditional HNCO, HNCA, HNCOCA and HNCACB-based 3D NMR spectroscopy. A 15 N-resolved TOCSY experiment has been used to assign Hα and Hβ chemical shifts.

The prevalence of intrinsically disordered protein regions, particularly in eukaryotic proteins, and their clear functional advantages for signaling and gene regulation have created an imperative for high-resolution structural and mechanistic studies. NMR spectroscopy has played a central role in enhancing not only our understanding of the intrinsically disordered native state, but also how that state contributes to biological function. While pathological functions associated with protein aggregation are well established, it has recently become clear that disordered regions also mediate functionally advantageous assembly into high-order structures that promote the formation of membrane-less sub-cellular compartments and even hydrogels. Across the range of functional assembly states accessed by disordered regions, post-translational modifications and regulatory macromolecular interactions, which can also be investigated by NMR spectroscopy, feature prominently. Here we will explore the many ways in which NMR has advanced our understanding of the physical-chemical phase space occupied by disordered protein regions and provide prospectus for the future role of NMR in this emerging and exciting field., (Copyright © 2017 Elsevier Inc. All rights reserved.)

This study investigated the associations between stressful family life events and adolescent externalizing and internalizing behaviors, and the interactive effects of family life events and cortisol reactivity on problem behaviors. In a sample of 100 mothers and their adolescents (M age = 15.09; SD age = .98; 68 % girls), adolescent cortisol reactivity was measured in response to a mother-adolescent conflict interaction task designed to elicit a stress response. Mothers reported on measures of family life events and adolescent problem behaviors. Results indicated that a heightened adolescent cortisol response moderated the relations between stressful family life events and both externalizing and internalizing behaviors. Results support context-dependent theoretical models, suggesting that for adolescents with higher cortisol reactivity (compared to those with lower cortisol reactivity), higher levels of stressful family life events were associated with greater problem behaviors, whereas lower levels of stressful family life events were related to fewer problem behaviors.

The gastrointestinal tract is continuously exposed to many environmental factors that influence intestinal epithelial cells and the underlying mucosal immune system. In this article, we demonstrate that dietary fiber and short chain fatty acids (SCFAs) induced the expression of the vitamin A-converting enzyme RALDH1 in intestinal epithelial cells in vivo and in vitro, respectively. Furthermore, our data showed that the expression levels of RALDH1 in small intestinal epithelial cells correlated with the activity of vitamin A-converting enzymes in mesenteric lymph node dendritic cells, along with increased numbers of intestinal regulatory T cells and a higher production of luminal IgA. Moreover, we show that the consumption of dietary fiber can alter the composition of SCFA-producing microbiota and SCFA production in the small intestines. In conclusion, our data illustrate that dietary adjustments affect small intestinal epithelial cells and can be used to modulate the mucosal immune system., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Objective: The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3 ) and β (LXRβ, NR1H2 ), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study., Approach and Results: We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease-exonuclease-phosphatase family domain containing 1 ( EEPD1/KIAA1706 ) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages., Conclusions: In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1., (© 2017 The Authors.)

Cellular cholesterol metabolism is subject to tight regulation to maintain adequate levels of this central lipid molecule. Herein, the sterol-responsive Liver X Receptors (LXRs) play an important role owing to their ability to reduce cellular cholesterol load. In this context, identifying the full set of LXR-regulated genes will contribute to our understanding of their role in cholesterol metabolism. Using global transcriptional analysis we report here the identification of RNF145 as an LXR-regulated target gene. We demonstrate that RNF145 is regulated by LXRs in both human and mouse primary cells and cell lines, and in vivo in mice. Regulation of RNF145 by LXR depends on a functional LXR-element in its proximal promotor. Consistent with LXR-dependent regulation of Rnf145 we show that regulation is lost in macrophages and fibroblasts from Lxrαβ(-/-) mice, and also in vivo in livers of Lxrα(-/-) mice treated with the LXR synthetic ligand T0901317. RNF145 is closely related to RNF139/TRC8, an E3 ligase implicated in control of SREBP processing. However, silencing of RNF145 in HepG2 or HeLa cells does not impair SREBP1/2 processing and sterol-responsive gene expression in these cells. Similar to TRC8, we demonstrate that RNF145 is localized to the ER and that it possesses intrinsic E3 ubiquitin ligase activity. In summary, we report the identification of RNF145 as an ER-resident E3 ubiquitin ligase that is transcriptionally controlled by LXR.

This study examined racial differences across African American, Hispanic, and White participants in the impact that individualand social risk factorshave on drinking behaviortrajectories.Data from the National Longitudinal Study of Adolescent Health was usedfrom 7-9 th graders (N = 4,372).Participants reported on frequency of drinking across the four waves and risk factors at wave 1. Growth mixture modeling revealed four trajectories for alcohol use that includedAbstainers, Early Starters, Late Starters, and De-Escalators. Social and individual indicators of risk were differently predictive of group membership to the problematic drinking trajectories. Differences across racial groups suggested that a lack of future orientation may be a salient risk factor for African Americanand Hispanicyouth's alcohol use, and peer alcohol use may be a salient risk factor for White youth's alcohol use. The findings of this study suggest that there may be individual differences in risk factors that provide insight for prevention efforts.

Calcineurin is a Ser/Thr phosphatase that is important for key biological processes, including immune system activation. We previously identified a region in the intrinsically disordered regulatory domain of calcineurin that forms a critical amphipathic α-helix (the "distal helix") that is required for complete activation of calcineurin. This distal helix was shown to have a Tm close to that of human body temperature. Because the Tm was determined in dilute buffer, we hypothesized that other factors inherent to a cellular environment might modulate the stability of the distal helix. One such factor that contributes to stability in other proteins is macromolecular crowding. The cell cytoplasm is comprised of up to 400 g/L protein, lipids, nucleic acids, and other compounds. We hypothesize that the presence of such crowders could increase the thermal stability of the distal helix and thus lead to a more robust activation of calcineurin in vivo. Using biophysical and biochemical approaches, we show that the distal helix of calcineurin is indeed stabilized when crowded by the synthetic polymers dextran 70 and ficoll 70, and that this stabilization of the distal helix increases the activity of calcineurin.

The Libbrecht-Hall circuit is a well-known, low-noise current driver for narrow-linewidth diode lasers. An important feature of the circuit is a current limit to protect the laser diode. As the current approaches the maximum limit, however, the noise in the laser current increases dramatically. This paper documents this behavior and explores simple circuit modifications to alleviate this issue.

Cholesterol metabolism is subject to complex transcriptional and nontranscriptional regulation. Herein, the role of ubiquitylation is emerging as an important post-translational modification that regulates cholesterol synthesis and uptake. Similar to other post-translational modifications, ubiquitylation is reversible in a process dependent on activity of deubiquitylating enzymes (DUBs). Yet whether these play a role in cholesterol metabolism is largely unknown. As a first step to test this possibility, we used pharmacological inhibition of cellular DUB activity. Short term (2 h) inhibition of DUBs resulted in accumulation of high molecular weight ubiquitylated proteins. This was accompanied by a dramatic decrease in abundance of the LDLR and attenuated LDL uptake into hepatic cells. Importantly, this occurred in the absence of changes in the mRNA levels of the LDLR or other SREBP2-regulated genes, in line with this phenotype being a post-transcriptional event. Mechanistically, we identify transcriptional induction of the E3 ubiquitin ligase IDOL in human and rodent cells as the underlying cause for ubiquitylation-dependent lysosomal degradation of the LDLR following DUB inhibition. In contrast to the established transcriptional regulation of IDOL by the sterol-responsive liver X receptor (LXR) transcription factors, induction of IDOL by DUB inhibition is LXR-independent and occurs in Lxrαβ(-/-) MEFs. Consistent with the role of DUBs in transcriptional regulation, we identified a 70-bp region in the proximal promoter of IDOL, distinct from that containing the LXR-responsive element, which mediates the response to DUB inhibition. In conclusion, we identify a sterol-independent mechanism to regulate IDOL expression and IDOL-mediated lipoprotein receptor degradation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cholesterol synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of cholesterol biosynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in cholesterol biosynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular cholesterol accretion that relies on uncoupling of cholesterol synthesis from lipoprotein uptake., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Developing autonomy and maintaining relatedness within the parent-adolescent relationship marks a realignment process that shifts adolescents' decision making and regulation from parents to youth. This process may be stressful for some adolescents, particularly those who perceive their daily lives as stressful. This study examined the associations of autonomy, relatedness and perceived stress with adolescents' cortisol and blood pressure response to conflict in a mother-adolescent interaction task among 100 adolescents (M age = 15.09; 68 % girls, 78 % Caucasian). Few direct associations were found, but results indicated that perceived stress moderated the effect of autonomy and relatedness such that youth who reported more perceived stress and whose mothers' restricted their autonomy and undermined their relatedness evidenced increased cortisol and systolic blood pressure when compared to youth lower in perceived stress. The results highlight the importance of examining individual differences in the association between normative developmental transitions and adolescents' neurobiological response to stress.

Adolescence is a unique period of heightened emotional arousal and still-developing regulatory abilities. Adolescent emotion regulation patterns may be critically involved in adolescents' psychosocial development, but patterns of emotion regulation in youths are not well understood. The current study used latent profile analysis (LPA) to elucidate patterns of emotion expression, experience, and emotion-related physiological arousal in adolescents. A sample of 198 adolescents and their primary caregivers participated in an emotionally arousing parent-adolescent conflict interaction. Adolescents' observed emotion expressions, emotion experiences, and heart rate (HR) and caregiver parenting behaviors were assessed during and/or after the interaction. Parents reported on adolescents' internalizing and externalizing symptoms, and youths reported on depressive symptoms. The LPA revealed four emotion regulation profiles: a moderate HR and high expression profile, a suppression profile (with low negative emotion expression and high emotion experience), a low reactive profile, and a high reactive profile. The moderate HR and high expression profile was associated with lower conduct disorder symptoms, the suppression profile was related to lower anxiety symptoms, and the high reactive profile was associated with higher adolescent depressive symptoms. The high reactive profile and moderate HR and high expression profile were associated with more negative/critical parenting behaviors. Findings suggest that profiles of adolescent emotion regulation can be empirically identified and may be significant risk factors for psychopathology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Identifying and then optimizing initial crystallization conditions is a prerequisite for macromolecular structure determination by crystallography. Improved technologies enable data collection on crystals that are difficult if not impossible to detect using visible imaging. The application of second-order nonlinear imaging of chiral crystals and ultraviolet two-photon excited fluorescence detection is shown to be applicable in a high-throughput manner to rapidly verify the presence of nanocrystals in crystallization screening conditions. It is noted that the nanocrystals are rarely seen without also producing microcrystals from other chemical conditions. A crystal volume optimization method is described and associated with a phase diagram for crystallization.

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN-DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS-induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA-mediated signaling is crucial for the efficiency of the mucosal immune system., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Latent transition analysis was used to identify patterns and trajectories of antisocial behavior (ASB) and their association with young adult outcomes in a nationally representative sample of adolescents (N = 5,422; 53.9% female). Participants were on average 13.96 years of age (SD = 1.06) at wave 1 of the study. Latent class analysis identified four classes of ASB including a non-ASB class, an aggressive class, a petty theft class, and a serious ASB class. In general, youth who were classified as serious stable ASB were the most at risk for problematic functioning in young adulthood. Youth who escalated to more serious patterns of ASB or reduced involvement also were at greater risk of negative outcomes in young adulthood compared to stable non-ASB youth, although they generally fared better than youth involved in stable patterns of more serious ASB. Gender differences indicated that involvement in ASB was a greater risk factor for alcohol use among boys and a greater risk factor for depression among girls in young adulthood. Results are discussed in terms of the predictive validity of classes of ASB to functioning in young adulthood and the implications of this research for prevention efforts.

An emerging theme in the regulation of cholesterol homeostasis is the role of the ubiquitin proteasome system (UPS), through which proteins are ubiquitylated and then degraded in response to specific signals. The UPS controls all aspects of cholesterol metabolism including its synthesis, uptake, and efflux. We review here recent work uncovering the ubiquitylation and degradation of key players in cholesterol homeostasis. This includes the low-density lipoprotein (LDL) receptor, transcription factors (sterol regulatory element binding proteins and liver X receptors), flux-controlling enzymes in cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase and squalene monooxygenase), and cholesterol exporters (ATP-binding cassette transporters ABCA1 and ABCG1). We explore which E3 ligases are involved, and identify areas deserving of further research., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.

Calcineurin (CaN) is a calmodulin-activated, serine/threonine phosphatase that is necessary for cardiac, vasculature, and nervous system development, as well as learning and memory, skeletal muscle growth, and immune system activation. CaN is activated in a manner similar to that of the calmodulin (CaM)-activated kinases. CaM binds CaN's regulatory domain (RD) and causes a conformational change that removes CaN's autoinhibitory domain (AID) from its catalytic site, activating CaN. In the CaM-activated kinases, the CaM binding region (CaMBR) is located just C-terminal to the AID, whereas in CaN, the AID is 52 residues C-terminal to the CaMBR. Previously published data have shown that these 52 residues in CaN's RD are disordered but approximately half of them gain structure, likely α-helical, upon CaM binding. In this work, we confirm that this increase in the level of structure is α-helical. We posit that this region forms an amphipathic helix upon CaM binding and folds onto the remainder of the RD:CaM complex, removing the AID. Förster resonance energy transfer data suggest the C-terminal end of this distal helix is relatively close to the N-terminal end of the CaMBR when the RD is bound by CaM. We show by circular dichroism spectroscopy and thermal melts that mutations on the hydrophobic face of the distal helix disrupt the structure gained upon CaM binding. Additionally, kinetic analysis of CaN activity suggests that these mutations affect CaN's ability to bind substrate, likely a result of the AID being able to bind to the active site even when CaM is bound. Our data demonstrate the presence of this distal helix and suggest it folds onto the remainder of the RD:CaM complex, creating a hairpinlike chain reversal that removes the AID from the active site.

Activated fibroblasts, also known as myofibroblasts, are mediators of several major human pathologies including proliferative fibrotic disorders, invasive tumor growth, rheumatoid arthritis, and atherosclerosis. We previously identified Niemann-Pick type C2 (NPC2) protein as a negative regulator of fibroblast activation (Csepeggi, C., Jiang, M., Kojima, F., Crofford, L. J., and Frolov, A. (2011) J. Biol. Chem. 286, 2078-2087). Here we report that NPC2-deficiency leads to a dramatic up-regulation of the arachidonic acid (AA) metabolic pathway in human fibroblasts. The major enzymes in this pathway, cPLA2 type IVA, COX-2, and mPGES-1, were dramatically up-regulated at both the transcriptional and translational levels. The specific phenotypic changes resulted in a >10-fold increase in the production and secretion of a key modulator of inflammation and immunity, prostaglandin E2. More importantly, AA metabolome profiling by liquid chromatography/tandem mass-spectrometry revealed the very specific nature of prostaglandin E2 up-regulation as the other analyzed AA metabolites derived from the COX-2, cytochrome P450, 5/15-lipoxygenase, and non-enzymatic oxidative pathways were mostly down-regulated. Blocking activity of cPLA2 efficiently suppressed expression of inflammatory cytokines, IL-1β and IL-6, thereby identifying cPLA2 as an important regulator of the inflammatory program in NPC2-null cells. Altogether, these studies highlight NPC2 as a specific regulator of AA metabolism and inflammation that suggests potential for NPC2 protein or its related signaling in the treatment of inflammatory diseases characterized by the presence of activated fibroblasts.

Objectives: We examined patterns of sexual behavior and risk for sexually transmitted infections (STIs) in young adulthood for Black, Hispanic, and White females., Methods: We used a nationally representative sample of 7015 female young adults from wave III of the National Longitudinal Study of Adolescent Health. Sexual risk items assessed behaviors occurring in the previous 6 years and past year to determine classes of sexual risk and links to STIs in young adulthood., Results: Latent class analysis revealed 3 sexual risk classes for Black and Hispanic youths and 4 sexual risk classes for White youths. The moderate and high risk classes had the highest probabilities of risky sexual partners, inconsistent condom use, and early age of sexual initiation, which significantly increased odds for STIs compared with recent abstainers., Conclusions: We found different classes of sexual behavior by race/ethnicity, with Black and Hispanic young women most at risk for STIs in young adulthood. Preventive efforts should target younger adolescents and focus on sexual partner behavior.

Adolescents' emotional reactivity in family, close friendships, and romantic relationships was examined in a community-based sample of 416 two-parent families. Six waves of annual data were analyzed using structural equation modeling. Emotional reactivity to interparental conflict during early adolescence was associated prospectively with adolescents' reactivity to conflict in friendships and romantic relationships during middle adolescence. Close friendship reactivity partially explained the prospective association between reactivity to interparental conflict and romantic relationship reactivity. The association between perceived emotional reactivity and relationship conflict was stronger for girls than boys. Results have important developmental implications regarding adolescents' emotional reactivity across salient interpersonal contexts during adolescence., ((c) 2013 APA, all rights reserved.)

The fungus Aspergillus fumigatus is a leading infectious killer in immunocompromised patients. Calcineurin, a calmodulin (CaM)-dependent protein phosphatase comprised of calcineurin A (CnaA) and calcineurin B (CnaB) subunits, localizes at the hyphal tips and septa to direct A. fumigatus invasion and virulence. Here we identified a novel serine-proline rich region (SPRR) located between two conserved CnaA domains, the CnaB-binding helix and the CaM-binding domain, that is evolutionarily conserved and unique to filamentous fungi and also completely absent in human calcineurin. Phosphopeptide enrichment and tandem mass spectrometry revealed the phosphorylation of A. fumigatus CnaA in vivo at four clustered serine residues (S406, S408, S410 and S413) in the SPRR. Mutation of the SPRR serine residues to block phosphorylation led to significant hyphal growth and virulence defects, indicating the requirement of calcineurin phosphorylation at the SPRR for its activity and function. Complementation analyses of the A. fumigatus ΔcnaA strain with cnaA homologs from the pathogenic basidiomycete Cryptococcus neoformans, the pathogenic zygomycete Mucor circinelloides, the closely related filamentous fungi Neurospora crassa, and the plant pathogen Magnaporthe grisea, revealed filamentous fungal-specific phosphorylation of CnaA in the SPRR and SPRR homology-dependent restoration of hyphal growth. Surprisingly, circular dichroism studies revealed that, despite proximity to the CaM-binding domain of CnaA, phosphorylation of the SPRR does not alter protein folding following CaM binding. Furthermore, mutational analyses in the catalytic domain, CnaB-binding helix, and the CaM-binding domains revealed that while the conserved PxIxIT substrate binding motif in CnaA is indispensable for septal localization, CaM is required for its function at the hyphal septum but not for septal localization. We defined an evolutionarily conserved novel mode of calcineurin regulation by phosphorylation in filamentous fungi in a region absent in humans. These findings suggest the possibility of harnessing this unique SPRR for innovative antifungal drug design to combat invasive aspergillosis.

This study examined the prospective relationship between negative parenting behaviors and adolescents' friendship competence in a community sample of 416 two-parent families in the Southeastern USA. Adolescents' externalizing problems and their emotional insecurity with parents were examined as mediators. Parents' psychological control was uniquely associated with adolescents' friendship competence. When both mediators were included in the same model, adolescents' perceptions of emotional insecurity in the parent-adolescent relationship fully mediated the association between parents' psychological control and adolescents' friendship competence. Parental hostility was associated with friendship competence indirectly through adolescents' emotional insecurity. Results contribute to identifying the mechanisms by which parenting affects youths' friendship competence, which is important in informing theory and practice regarding interpersonal relationships in adolescence.

Experience with and management of stress has implications for adolescents' behavioral and socioemotional development. This study examined the relationship between adolescents' physiological response to an acute laboratory stressor (i.e., Trier Social Stress Test; TSST) and anger regulation and interpersonal competence in a sample of 175 low-income urban adolescents (51.8% girls). Findings suggested that heightened reactivity as indicated by cortisol, heart rate, and blood pressure was associated with increased interpersonal competence and anger regulation. However, these findings were context dependent such that, for youth high in self-reported child maltreatment, heightened reactivity was associated with decreased interpersonal competence and anger regulation. Results highlight the importance of considering how context may condition the effect of stress reactivity on functioning during adolescence.