Comparative analysis of thermal adaptations of extremophilic prolyl oligopeptidases.

Prolyl oligopeptidases from psychrophilic, mesophilic, and thermophilic organisms found in a range of natural environments are studied using a combination of protein structure prediction, atomistic molecular dynamics, and trajectory analysis to determine how the S9 protease family adapts to extreme thermal conditions. We compare our results with hypotheses from the literature regarding structural adaptations that allow proteins to maintain structure and function at extreme temperatures, and we find that, in the case of prolyl oligopeptidases, only a subset of proposed adaptations are employed for maintaining stability. The catalytic and propeller domains are highly structured, limiting the range of mutations that can be made to enhance hydrophobicity or form disulfide bonds without disrupting the formation of necessary secondary structure. Rather, we observe a pattern in which overall prevalence of bound interactions (salt bridges and hydrogen bonds) is conserved by using increasing numbers of increasingly short-lived interactions as temperature increases. This suggests a role for an entropic rather than energetic strategy for thermal adaptation in this protein family.

Mutagenesis on a complex mouse genetic background by site-specific nucleases.

Mouse models with complex genetic backgrounds are increasingly used in preclinical research to accurately model human disease and to enable temporal and cell-specific evaluation of genetic manipulations. Backcrossing mice onto these complex genetic backgrounds takes time and leads to significant wastage of animals. In this study, we aimed to evaluate whether site-specific nucleases could be used to generate additional genetic mutations in a complex genetic background, using the REVERSA mouse model of atherosclerosis, a model harbouring four genetically altered alleles. The model is comprised of a functional null mutation in the Ldlr gene in combination with a ApoB100 allele, which, after high-fat diet, leads to the rapid development of atherosclerosis. The regression of the pathology is achieved by inducible knock-out of the Mttp gene. Here we report an investigation to establish if microinjection of site-specific nucleases directly into zygotes prepared from the REVERSA could be used to investigate the role of the ATP binding cassette transporter G1 (ABCG1) in atherosclerosis regression. We show that using this approach we could successfully generate two independent knockout lines on the REVERSA background, both of which exhibited the expected phenotype of a significant reduction in cholesterol efflux to HDL in bone marrow-derived macrophages. However, loss of Abcg1 did not impact atherosclerosis regression in either the aortic root or in aortic arch, demonstrating no important role for this transporter subtype. We have demonstrated that site-specific nucleases can be used to create genetic modifications directly onto complex disease backgrounds and can be used to explore gene function without the need for laborious backcrossing of independent strains, conveying a significant 3Rs advantage.

Mutation Effects on Structure and Dynamics: Adaptive Evolution of the SARS-CoV-2 Main Protease.

The main protease of SARS-CoV-2 (Mpro) plays a critical role in viral replication; although it is relatively conserved, Mpro has nevertheless evolved over the course of the COVID-19 pandemic. Here, we examine phenotypic changes in clinically observed variants of Mpro, relative to the originally reported wild-type enzyme. Using atomistic molecular dynamics simulations, we examine effects of mutation on protein structure and dynamics. In addition to basic structural properties such as variation in surface area and torsion angles, we use protein structure networks and active site networks to evaluate functionally relevant characters related to global cohesion and active site constraint. Substitution analysis shows a continuing trend toward more hydrophobic residues that are dependent on the location of the residue in primary, secondary, tertiary, and quaternary structures. Phylogenetic analysis provides additional evidence for the impact of selective pressure on mutation of Mpro. Overall, these analyses suggest evolutionary adaptation of Mpro toward more hydrophobicity and a less-constrained active site in response to the selective pressures of a novel host environment.

The importance of high-quality partner support for reducing stress during pregnancy and postpartum bonding impairments.

The current study investigated the extent to which interparental support reduced pregnancy stress and subsequent postpartum bonding impairments with infant. We hypothesized that receiving higher quality partner support would be associated with decreased maternal pregnancy-related concerns, and less maternal and paternal pregnancy stress which, in turn, would predict fewer parent-infant bonding impairments. One hundred fifty-seven cohabiting couples completed semi-structured interviews and questionnaires once during pregnancy and twice postpartum. Path analyses with tests of mediation were employed to test our hypotheses. Higher quality support received by mothers was associated with lower maternal pregnancy stress which, in turn, predicted fewer mother-infant bonding impairments. An indirect pathway of equal magnitude was observed for fathers. Dyadic pathways also emerged such that higher quality support received by fathers was associated with lower maternal pregnancy stress which reduced mother-infant bonding impairments. Similarly, higher quality support received by mothers reduced paternal pregnancy stress and subsequent father-infant bonding impairments. Hypothesized effects reaching statistical significance (p < .05) were small to moderate in magnitude. These findings have important theoretical and clinical implications in demonstrating the critical role of both receiving and providing high-quality interparental support to reduce pregnancy stress and subsequent postpartum bonding impairments for mothers and fathers. Results also highlight the utility of investigating maternal mental health in the couple context.

α-Crystallins in the Vertebrate Eye Lens: Complex Oligomers and Molecular Chaperones.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged ß- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.

A preliminary assessment of the link between anxiety sensitivity cognitive concerns and suicidal thoughts through dissociative symptoms.

Anxiety sensitivity (AS) and AS subfactors (physical, cognitive, and social) have been found to have robust associations with suicide risk. While the direct association between AS subfactors and suicide risk have been explored, little is known about how specific mechanisms, such as dissociation, might explain this relationship. This study aimed to run three analyses to examine the direct and indirect effects of suicidal thoughts and AS via dissociative symptoms. We predicted that dissociation would be a pathway through which AS physical concerns (ASPC) and AS cognitive concerns (ASCC) predicts suicidal ideation. Participants included 84 undergraduate students from a Southeastern University who were elevated on ASCC. Participants completed measures examining dissociative experiences, anxiety sensitivity, and current suicidal ideation. Results revealed that dissociation had a significant indirect effect with ASPC but not ASCC. The current preliminary study showed that ASCC had direct associations with suicide risk; however, those with lower levels of ASPC and dissociation may also be more likely to develop suicide risk. Future research should explore the possibility that the dissociation/ASPC and ASCC pathways are separate, but related, paths to suicidality.

Combining virtual reality and 3D-printed models to simulate patient-specific dental operative procedures-A study exploring student perceptions.

INTRODUCTION: Students face a number of challenges in translating the skills acquired in pre-clinical simulation environments to the delivery of real patient care. These are particularly emphasised for complex operative procedures such as tooth preparations for indirect restorations. This paper reports student perceptions of a novel approach designed to improving student confidence when undertaking operative procedures on patients for the first time, by providing patient-specific simulation using virtual reality (VR) and 3D-printed models of the student's real clinical case. MATERIALS AND METHODS: Students practised on patient-specific models, in the presence of a clinical tutor, firstly using VR simulation then with 3D-printed models in a clinical skills laboratory. The students then carried out the operative procedure on their patients, on the third occasion of practice. After providing the treatment for their patients, students attended a semi-structured interview to discuss their experiences. The qualitative data were analysed using two forms of inductive analysis. RESULTS: Students most frequently cited: the value of the educator, increased confidence and efficiency during the clinical procedure, improved patient confidence and the complementary benefits of the two simulation modalities. Thematic analysis of participants' responses uncovered five key themes: The value of virtual reality dental simulators The value of clinical skills laboratory simulation with 3D-printed models The value of educator engagement The impact on the clinical procedure and the patient The VR and clinical skills laboratory balance CONCLUSION: This paper reports the early findings of an intervention that improves dental student confidence through the use of patient-specific VR exercises and 3D-printed models. These provided an incremental learning experience for an operative clinical procedure, prior to treatment of the live patient. Early results suggest this is a positive experience for the students, providing a valuable contribution to their confidence and preparedness.

Chemical Properties Determine Solubility and Stability in ßγ-Crystallins of the Eye Lens.

ßγ-Crystallins are the primary structural and refractive proteins found in the vertebrate eye lens. Because crystallins are not replaced after early eye development, their solubility and stability must be maintained for a lifetime, which is even more remarkable given the high protein concentration in the lens. Aggregation of crystallins caused by mutations or post-translational modifications can reduce crystallin protein stability and alter intermolecular interactions. Common post-translational modifications that can cause age-related cataracts include deamidation, oxidation, and tryptophan derivatization. Metal ion binding can also trigger reduced crystallin solubility through a variety of mechanisms. Interprotein interactions are critical to maintaining lens transparency: crystallins can undergo domain swapping, disulfide bonding, and liquid-liquid phase separation, all of which can cause opacity depending on the context. Important experimental techniques for assessing crystallin conformation in the absence of a high-resolution structure include dye-binding assays, circular dichroism, fluorescence, light scattering, and transition metal FRET.

Function and Aggregation in Structural Eye Lens Crystallins.

Crystallins are transparent, refractive proteins that contribute to the focusing power of the vertebrate eye lens. These proteins are extremely soluble and resist aggregation for decades, even under crowded conditions. Crystallins have evolved to avoid strong interprotein interactions and have unusual hydration properties. Crystallin aggregation resulting from mutation, damage, or aging can lead to cataract, a disease state characterized by opacity of the lens.Different aggregation mechanisms can occur, following multiple pathways and leading to aggregates with varied morphologies. Studies of variant proteins found in individuals with childhood-onset cataract have provided insight into the molecular factors underlying crystallin stability and solubility. Modulation of exposed hydrophobic surface is critical, as is preventing specific intermolecular interactions that could provide nucleation sites for aggregation. Biophysical measurements and structural biology techniques are beginning to provide a detailed picture of how crystallins crowd into the lens, providing high refractivity while avoiding excessively tight binding that would lead to aggregation.Despite the central biological importance of refractivity, relatively few experimental measurements have been made for lens crystallins. Our work and that of others have shown that hydration is important to the high refractive index of crystallin proteins, as are interactions between pairs of aromatic residues and potentially other specific structural features.This Account describes our efforts to understand both the functional and disease states of vertebrate eye lens crystallins, particularly the γ-crystallins. We use a variety of biophysical techniques, notably NMR spectroscopy, to investigate crystallin stability and solubility. In the first section, we describe efforts to understand the relative stability and aggregation propensity of different γS-crystallin variants. The second section focuses on interactions of these proteins with the holdase chaperone αB-crystallin. The third, fourth, and fifth sections explore different modes of aggregation available to crystallin proteins, and the final section highlights the importance of refractive index and the sometimes conflicting demands of selection for refractivity and solubility.

Disruption of global hypothalamic microRNA (miR) profiles and associated behavioral changes in California mice (Peromyscus californicus) developmentally exposed to endocrine disrupting chemicals.

Developmental exposure to endocrine disrupting chemicals (EDCs), e.g., bisphenol A (BPA) or genistein (GEN), causes longstanding epigenome effects. MicroRNAs (miRs) regulate which mRNAs will be translated to proteins and thereby serve as the final checkpoint in epigenetic control. Scant amount is known, however, whether EDCs affect neural miRNA (miR) patterns. We aimed to test the hypothesis that developmental exposure of California mice (Peromyscus californicus) to GEN, BPA, or both chemicals influences hypothalamic miR/small RNA profiles and ascertain the extent such biomolecular alterations correlate with behavioral and metabolic changes. California mice were developmentally exposed to GEN (250 mg/kg feed weight, FW), GEN (250 mg/kg FW)+BPA (5 mg/kg FW), low dose (LD) BPA (5 mg/kg FW), or upper dose (UD) BPA (50 mg/kg FW). Adult offspring were tested in a battery of behavioral and metabolic tests; whereupon, mice were euthanized, brains were collected and frozen, small RNAs were isolated from hypothalamic punches, and subsequently sequenced. California mice exposed to one or both EDCs engaged in one or more repetitive behaviors. GEN, LD BPA, and UD BPA altered aspects of ultrasonic and audible vocalizations. Each EDC exposure led to sex-dependent differences in differentially expressed miR/small RNAs with miR7-2, miR146, and miR148a being increased in all female and male EDC exposed groups. Current findings reveal that developmental exposure to GEN and/or BPA affects hypothalamic miR/small RNA expression patterns, and such changes correlate with EDC-induced behavioral and metabolic alterations. miR146 is likely an important mediator and biomarker of EDC exposure in mammals, including humans.

Impact of pertussis-specific IgA, IgM, and IgG antibodies in mother's own breast milk and donor breast milk during preterm infant digestion.

BACKGROUND: The survival of antibody isotypes specific to pertussis toxin (PT) and filamentous hemagglutinin (FHA) from mother's own milk (MBM) and donor breast milk (DBM) during preterm infant digestion was investigated. METHODS: Feed, gastric, and stool samples were collected from 20 preterm mother-infant pairs at 8-9 days and 21-22 days postpartum. Samples were analyzed via ELISA for anti-FHA or anti-PT immunoglobulin A (IgA), IgM, and IgG. RESULTS: Anti-PT IgA, anti-FHA IgG, and anti-PT IgG were lower in MBM than DBM at 8-9 days postpartum, whereas anti-FHA IgM was higher in MBM than DBM. Anti-PT IgA, anti-PT IgG, and anti-FHA IgG in DBM decreased in gastric contents at both postpartum times but those antibodies in MBM were stable or increased during gastric digestion. Anti-FHA-specific IgA and IgM were higher in gastric contents from infants fed MBM than from infants fed DBM at 8-9 days. All pertussis antibodies were detected in infant stools at both postpartum times. CONCLUSIONS: Pertussis-specific antibodies from MBM were stable during infant digestion, whereas anti-pertussis IgA and IgG from DBM decreased in gastric contents. The constant region and variable region of antibodies and maternal immunization appear to be the critical factors for their stability to proteolytic digestion and pasteurization. IMPACT: Pertussis-specific antibodies from mother's breast milk were stable during infant digestion, whereas anti-pertussis IgA and IgG from donor breast milk decreased in gastric contents. The constant region and variable region of pertussis-specific antibodies and the maternal immunization (previous infections and vaccinations) appear to be the critical factors for their stability to proteolytic digestion and pasteurization. Pertussis-specific antibodies from either mother's breast milk or donor breast milk survived during preterm infant digestion and both types of milk will compensate for the lower IgG transplacental transfer in preterm infants compared with term infants.

Bayesian estimation of the hydroxyl radical diffusion coefficient at low temperature and high pressure from atomistic molecular dynamics.

The hydroxyl radical is the primary reactive oxygen species produced by the radiolysis of water and is a significant source of radiation damage to living organisms. Mobility of the hydroxyl radical at low temperatures and/or high pressures is hence a potentially important factor in determining the challenges facing psychrophilic and/or barophilic organisms in high-radiation environments (e.g., ice-interface or undersea environments in which radiative heating is a potential heat and energy source). Here, we estimate the diffusion coefficient for the hydroxyl radical in aqueous solution using a hierarchical Bayesian model based on atomistic molecular dynamics trajectories in TIP4P/2005 water over a range of temperatures and pressures.

'It's been adapted rather than impacted': A qualitative evaluation of the impact of Covid-19 restrictions on the positive behavioural support of people with an intellectual disability and/or autism.

BACKGROUND: We used a qualitative approach to explore the experiences of social care staff regarding the provision of positive behavioural support (PBS) to people with an intellectual disability at the height of the Covid-19 restrictions. METHOD: We conducted semi-structured interviews with 19 staff who had recently completed a PBS workforce development programme. Data were analysed using thematic analysis. RESULTS: Three themes were identified in the context of the restrictions: The challenges to maintaining quality of life and PBS of the people being supported and staff attempts to overcome these; the ways in which PBS and behaviour support plans were implemented and the impact on behaviours that challenge; the ways in which PBS principles were applied at organisational levels to help to understand and address staff stress and distress. CONCLUSIONS: Overall, the staff identified many unexpected benefits of the restrictions. The results are discussed in the context of the study limitations.

A quantitative evaluation of a regional Positive Behavioural Support workforce development approach.

BACKGROUND: Research suggests that providing staff with input in relation to Positive Behavioural Support (PBS) can have beneficial outcomes. Much of this research, however, fails to take account of systemic issues and does not include a control group. METHOD: We used a non-randomised, controlled group design to evaluate accredited PBS programmes, delivered as part of a systemic, regional and workforce development approach. We compared outcomes of those attending the programmes (n = 240) with a control group (n = 54), pre- and post-intervention and at 3-months follow-up. RESULTS: The programme and its wider impact were rated positively. Significant intervention effects were found for staff practice and retention, but not for staff knowledge and attributions, or behaviours that challenge and quality of life of those being supported. CONCLUSIONS: The results are discussed in the context of the study limitations and restrictions resulting from the Covid-19 pandemic.

Sequence Characterization and Molecular Modeling of Clinically Relevant Variants of the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro) is essential to viral replication and cleaves highly specific substrate sequences, making it an obvious target for inhibitor design. However, as for any virus, SARS-CoV-2 is subject to constant neutral drift and selection pressure, with new Mpro mutations arising over time. Identification and structural characterization of Mpro variants is thus critical for robust inhibitor design. Here we report sequence analysis, structure predictions, and molecular modeling for seventy-nine Mpro variants, constituting all clinically observed mutations in this protein as of April 29, 2020. Residue substitution is widely distributed, with some tendency toward larger and more hydrophobic residues. Modeling and protein structure network analysis suggest differences in cohesion and active site flexibility, revealing patterns in viral evolution that have relevance for drug discovery.

Human γS-Crystallin-Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

Milk Peptides Survive In Vivo Gastrointestinal Digestion and Are Excreted in the Stool of Infants.

BACKGROUND: Human milk peptides released by gastrointestinal proteases have been identified with bioactivities that can benefit the infant but must first reach their respective sites of activity. Peptides in the stool either survived to or were released inside the intestinal tract, and thus had the opportunity to exert bioactivity there. However, it is unknown whether any milk peptides, bioactive or not, can survive in the stool of infants. OBJECTIVE: The aim of this study was primarily to identify milk peptides in infant stool samples and secondarily test the hypotheses that the milk peptide profiles of stools are different between preterm infants at different days of life and between preterm and term infants. METHODS: Infant stool samples were collected from 16 preterm infants (<34 weeks gestational age) at 8 or 9 and 21 or 22 days of life (DOL), and from 10 term infants (>34 weeks gestational age) at 8 or 9 DOL. Milk peptides were isolated from the stool samples and identified using tandem MS. The peptide counts and abundances were compared between infant groups. RESULTS: In total, 118 exclusively milk-derived peptides from the caseins and α-lactalbumin were present in the stool samples, including some peptides with known or potential bioactivity. The remaining 8014 identified peptides could be derived either from milk or endogenous proteins. Although many individual milk peptides were significantly different between preterm infants at 8/9 and 21/22 DOL and between preterm and term infants, total peptide abundance and count were similar for all 3 groups. CONCLUSIONS: This is the first study to confirm the survival of milk peptides in the stool of infants. Some of the peptides had potential bioactivities that could influence infant gut development. These results are important to understand the physiological relevance of human milk peptides to the infant.

Talking about Decisions: The Facilitating Effect of a Celebrity Health Announcement on the Communication of Online Personal Experiences around Decision-making.

This study examines how posters on an online forum discussed their own current and past decision-making in response to a celebrity health announcement. We examined messages in response to a celebrity narrative, and extracted those explicitly related to decision-making around genetic testing and preventative surgery. Using deductive coding we identified different types of decision-making narratives, and with inductive coding we examined how users discussed decision-making activities. Guided by the literature on narrative content types in decision-making, and on celebrity health narratives, we found that the celebrity decision announcement facilitated social sharing in relation to two key decision-making activities. First, identifying with the celebrity allowed people to reflect and compare their own personal health circumstances. This empowered readers to appraise and select options about their current decision-making regarding preventative surgery. Second, the announcement allowed an extension of the discussion beyond the celebrity, and acted as a catalyst encouraging other people to share their own previous decision-making experiences. These experiences contained a mix of narratives content types, and provided an opportunity for posters to evaluate their decisions, and to contribute to a repository of decision-making examples for others. Health narratives act as communication devices in decision-making, and we discuss the findings in relation to the extension of the educational and persuasive function of celebrity health narratives.

An exploration of the factors influencing career choice in mental health.

AIMS AND OBJECTIVES: To identify the factors that are associated with considering a career in mental health. BACKGROUND: The mental health specialty is facing a recruitment crisis in the United Kingdom but there is limited evidence about which factors encourage and discourage people from considering a career in mental health. DESIGN: Quantitative, observational, online survey using a multiple ordinal logistic regression model to identify if there were any significant predictors of the extent to which participants would consider a career in mental health. The design and write up of the study were guided by the STROBE checklist. METHOD: We gathered the views of 231 participants (female = 188, 81.7%) aged between 16-65 (mean = 22.7, SD = 8.9), using an online survey, the majority of whom were studying on, or graduates of, psychology/social studies degrees. Information was gathered about the extent to which a range of factors influenced consideration of a career in mental health. RESULTS: The majority (71.2%) of participants reported that they would definitely or probably consider undertaking a career in mental health, and over half (51.4%) would consider a career as a mental health nurse. The ability to help others and receiving appropriate training required for the role were important career choice factors. Being female, having a mental health condition and greater knowledge of mental health were associated with a significantly greater likelihood of considering a career in mental health, while having had experience of working with people with mental health difficulties was significantly negatively associated. CONCLUSIONS: Students and graduates of psychology and social studies degrees appear to be a large, untapped recruitment pool for mental health services. RELEVANCE TO CLINICAL PRACTICE: The results can inform more targeted recruitment strategies and development of suitable career pathways for those interested in a career in mental health.

The impact of an adult intellectual disability screening questionnaire on service providers and users.

BACKGROUND: One contributor to the health inequalities that people with an intellectual disability face is failure to identify their intellectual disability. The Learning Disability Screening Questionnaire (LDSQ) can identify adults who are likely to have an intellectual disability, but little is known about its impact. METHODS: A modified Delphi approach (literature search, interviews with staff and those using services [n = 28], and completion of an online survey by professionals [n = 29]) was used to develop a framework to evaluate the impact of the LDSQ. RESULTS: Items endorsed by 60% or more of respondents (9/18) were included in the final framework. These all related to benefits of the LDSQ, including identifying people not previously known to have an intellectual disability; helping prioritize diagnostic assessment; informing support needs; and helping increase wellbeing and life chances. CONCLUSION: The LDSQ may offer one way of helping address the health inequalities that people with an intellectual disability face.