New living evidence resource of human and non-human studies for early intervention and research prioritisation in anxiety, depression and psychosis.

In anxiety, depression and psychosis, there has been frustratingly slow progress in developing novel therapies that make a substantial difference in practice, as well as in predicting which treatments will work for whom and in what contexts. To intervene early in the process and deliver optimal care to patients, we need to understand the underlying mechanisms of mental health conditions, develop safe and effective interventions that target these mechanisms, and improve our capabilities in timely diagnosis and reliable prediction of symptom trajectories. Better synthesis of existing evidence is one way to reduce waste and improve efficiency in research towards these ends. Living systematic reviews produce rigorous, up-to-date and informative evidence summaries that are particularly important where research is emerging rapidly, current evidence is uncertain and new findings might change policy or practice. Global Alliance for Living Evidence on aNxiety, depressiOn and pSychosis (GALENOS) aims to tackle the challenges of mental health science research by cataloguing and evaluating the full spectrum of relevant scientific research including both human and preclinical studies. GALENOS will also allow the mental health community-including patients, carers, clinicians, researchers and funders-to better identify the research questions that most urgently need to be answered. By creating open-access datasets and outputs in a state-of-the-art online resource, GALENOS will help identify promising signals early in the research process. This will accelerate translation from discovery science into effective new interventions for anxiety, depression and psychosis, ready to be translated in clinical practice across the world.

Staphylococcal Periscope proteins Aap, SasG, and Pls project noncanonical legume-like lectin adhesin domains from the bacterial surface.

Staphylococcus aureus and Staphylococcus epidermidis are frequently associated with medical device infections that involve establishment of a bacterial biofilm on the device surface. Staphylococcal surface proteins Aap, SasG, and Pls are members of the Periscope Protein class and have been implicated in biofilm formation and host colonization; they comprise a repetitive region ("B region") and an N-terminal host colonization domain within the "A region," predicted to be a lectin domain. Repetitive E-G5 domains (as found in Aap, SasG, and Pls) form elongated "stalks" that would vary in length with repeat number, resulting in projection of the N-terminal A domain variable distances from the bacterial cell surface. Here, we present the structures of the lectin domains within A regions of SasG, Aap, and Pls and a structure of the Aap lectin domain attached to contiguous E-G5 repeats, suggesting the lectin domains will sit at the tip of the variable length rod. We demonstrate that these isolated domains (Aap, SasG) are sufficient to bind to human host desquamated nasal epithelial cells. Previously, proteolytic cleavage or a deletion within the A domain had been reported to induce biofilm formation; the structures suggest a potential link between these observations. Intriguingly, while the Aap, SasG, and Pls lectin domains bind a metal ion, they lack the nonproline cis peptide bond thought to be key for carbohydrate binding by the lectin fold. This suggestion of noncanonical ligand binding should be a key consideration when investigating the host cell interactions of these bacterial surface proteins.

Trace amine-associated receptor 1 (TAAR1) agonists for psychosis: protocol for a living systematic review and meta-analysis of human and non-human studies.

BACKGROUND: There is an urgent need to develop more effective and safer antipsychotics beyond dopamine 2 receptor antagonists. An emerging and promising approach is TAAR1 agonism. Therefore, we will conduct a living systematic review and meta-analysis to synthesize and triangulate the evidence from preclinical animal experiments and clinical studies on the efficacy, safety, and underlying mechanism of action of TAAR1 agonism for psychosis. METHODS: Independent searches will be conducted in multiple electronic databases to identify clinical and animal experimental studies comparing TAAR1 agonists with licensed antipsychotics or other control conditions in individuals with psychosis or animal models for psychosis, respectively. The primary outcomes will be overall psychotic symptoms and their behavioural proxies in animals. Secondary outcomes will include side effects and neurobiological measures. Two independent reviewers will conduct study selection, data extraction using predefined forms, and risk of bias assessment using suitable tools based on the study design. Ontologies will be developed to facilitate study identification and data extraction. Data from clinical and animal studies will be synthesized separately using random-effects meta-analysis if appropriate, or synthesis without meta-analysis. Study characteristics will be investigated as potential sources of heterogeneity. Confidence in the evidence for each outcome and source of evidence will be evaluated, considering the summary of the association, potential concerns regarding internal and external validity, and reporting biases. When multiple sources of evidence are available for an outcome, an overall conclusion will be drawn in a triangulation meeting involving a multidisciplinary team of experts. We plan trimonthly updates of the review, and any modifications in the protocol will be documented. The review will be co-produced by multiple stakeholders aiming to produce impactful and relevant results and bridge the gap between preclinical and clinical research on psychosis. PROTOCOL REGISTRATION: PROSPERO-ID: CRD42023451628.

Diverse functions for acyltransferase-3 proteins in the modification of bacterial cell surfaces.

The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins - acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O-acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.

Relational interventions for organizational learning: An experience report.

INTRODUCTION: Quality improvement and implementation science practitioners identify relational issues as important obstacles to success. Relational interventions may be important for successful performance improvement and fostering Learning Health Systems. METHODS: This case report describes the experience and lessons learned from implementing a relational approach to organizational change, informed by Relational Coordination Theory, in a health system. Structured interviews were used to obtain qualitative participant feedback. Relational Coordination was measured serially using a validated seven-item survey. RESULTS: An initial, relational intervention on one unit promoted increased participant engagement, self-efficacy, and motivation that led to the spontaneous, emergent dissemination of relational change, and learning into other parts of the health system. Staff involved in the intervention reported increased systems thinking, enhanced focus on communication and relationships as key drivers for improvement and learning, and greater awareness of organizational change as something co-created by staff and executives. CONCLUSIONS: This experience supports the hypothesis that relational interventions are important for fostering the development of Learning Health Systems.

Periscope Proteins are variable-length regulators of bacterial cell surface interactions.

Changes at the cell surface enable bacteria to survive in dynamic environments, such as diverse niches of the human host. Here, we reveal "Periscope Proteins" as a widespread mechanism of bacterial surface alteration mediated through protein length variation. Tandem arrays of highly similar folded domains can form an elongated rod-like structure; thus, variation in the number of domains determines how far an N-terminal host ligand binding domain projects from the cell surface. Supported by newly available long-read genome sequencing data, we propose that this class could contain over 50 distinct proteins, including those implicated in host colonization and biofilm formation by human pathogens. In large multidomain proteins, sequence divergence between adjacent domains appears to reduce interdomain misfolding. Periscope Proteins break this "rule," suggesting that their length variability plays an important role in regulating bacterial interactions with host surfaces, other bacteria, and the immune system.

Informing National Health Service patients about participation in clinical research: A comparison of opt-in and opt-out approaches across the United Kingdom.

OBJECTIVE: Recruitment to clinical research in the National Health Service remains challenging. One barrier is accessing patients to discuss research participation. Two general approaches are used in the United Kingdom to facilitate this: an 'opt-in' approach (when clinicians communicate research opportunities to patients) and an 'opt-out' approach (all patients have the right to be informed of relevant research opportunities). No evidence-based data are available, however, to inform the decision about which approach is preferable. This study aimed to collect information from 'opt-in' and 'opt-out' Trusts and identify which of the two approaches is optimal for ensuring National Health Service patients are given opportunities to discuss research participation. METHOD: This sequential mixed methods study comprised three phases: (1) an Appreciative Inquiry across UK Trusts, (2) online surveys and (3) focus groups with National Health Service staff and patients at a representative mental health Trust. RESULTS: The study was conducted between June and October 2019. Out of seven National Health Service Mental Health Trusts contacted (three 'opt-out' and four 'opt-in'), only four took part in phase 1 of the study and three of them were 'opt-out' Trusts. Benefits of an 'opt-out' approach included greater inclusivity of patients and the removal of research gatekeepers, while the involvement of research-active clinicians and established patient-clinician relationships were cited as important to 'opt-in' success. Phases 2 and 3 were conducted at a different Trust (Oxford Health NHS Foundation Trust) which was using an 'opt-in' approach. Of 333 staff and member survey responders, 267 (80.2%) favoured moving to an 'opt-out' approach (phase 2). Nineteen staff and 16 patients and carers participated in focus groups (phase 3). Concern was raised by staff regarding the lack of time for clinical research, with clinical work taking precedence over research; patients were concerned about a lack of research activity; all considered research to be beneficial and were supportive of a move to 'opt-out'. CONCLUSION: Findings suggest that 'opt-out' is more beneficial than 'opt-in', with the potential to vastly increase patient access to research opportunities and to enable greater equality of information provision for currently marginalised groups. This should ensure that healthcare research is more representative of the entire population, including those with a mental health diagnosis.

Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase.

Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an "AT3-SGNH fused" domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in O-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates.

Rational Design and Self-Assembly of Coiled-Coil Linked SasG Protein Fibrils.

Protein engineering is an attractive approach for the self-assembly of nanometer-scale architectures for a range of potential nanotechnologies. Using the versatile chemistry provided by protein folding and assembly, coupled with amino acid side-chain functionality, allows for the construction of precise molecular "protein origami" hierarchical patterned structures for a range of nanoapplications such as stand-alone enzymatic pathways and molecular machines. The Staphyloccocus aureus surface protein SasG is a rigid, rod-like structure shown to have high mechanical strength due to "clamp-like" intradomain features and a stabilizing interface between the G5 and E domains, making it an excellent building block for molecular self-assembly. Here we characterize a new two subunit system composed of the SasG rod protein genetically conjugated with de novo designed coiled-coils, resulting in the self-assembly of fibrils. Circular dichroism (CD) and quartz-crystal microbalance with dissipation (QCM-D) are used to show the specific, alternating binding between the two subunits. Furthermore, we use atomic force microscopy (AFM) to study the extent of subunit polymerization in a liquid environment, demonstrating self-assembly culminating in the formation of linear macromolecular fibrils.

Consent to discuss participation in research: a pilot study.

BACKGROUND: Equitable access to research studies needs to be increased for all patients. There is debate about which is the best approach to use to discuss participation in research in real-world clinical settings. OBJECTIVE: We aimed to determine the feasibility of asking all clinical staff within one hospital Trust (an organisation that provides secondary health services within the English and Welsh National Health Service) to use a newly created form on the Trust's electronic patient records system, as a means of asking patients to consent to discuss participation in research (the opt-in approach). We also aimed to collect feedback from patients and clinicians about their views of the opt-in approach. METHODS: Four pilot sites were selected in the Trust: two memory clinics, an adult mental health team and an acute adult ward. Data were collected in three phases: (1) for 6 months, pilot site staff were asked to complete a consent to discuss participation in research form with patients; (2) staff feedback on the form was collected through an online survey; and (3) patient feedback was collected through focus groups. FINDINGS: Of 1779 patients attending services during the pilot period, 197 (11%) had a form completed by staff and 143 (8%) opted-in to finding out about research. Staff cited limited time, low priority and poor user experience of the electronic patient records system as reasons for low uptake of the form. Patients generally approved of the approach but offered suggestions for improvement. CONCLUSIONS: There were mixed results for adopting an opt-in approach; uptake was very low, limiting its value as an effective strategy for improving access to research. CLINICAL IMPLICATIONS: Alternative strategies to the opt-in approach, such as transparent opt out approaches, warrant consideration to maximise access to research within routine clinical care.

Ketamine for suicidal ideation in adults with psychiatric disorders: A systematic review and meta-analysis of treatment trials.

OBJECTIVE: Ketamine may reduce suicidal ideation in treatment-resistant depression. But it is not known how quickly this occurs and how long it persists. We undertook a systematic review and meta-analysis to determine the short- and long-term effectiveness of ketamine for suicidality. METHOD: CENTRAL, EMBASE, Medline, and PsycINFO were searched until 12 December 2018. Randomised controlled trials of ketamine or esketamine reporting data on suicidal ideation, self-harm, attempted or completed suicide in adults diagnosed with any psychiatric disorder were included. Two reviewers independently extracted data, and certainty of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation tool. Standardised mean difference was used for continuous outcomes. RESULTS: Twenty-five reports from 15 independent trials, with a total of 572 participants diagnosed with predominately affective disorders, were included. The evidence was rated moderate to low. In most trials, ketamine was administered at 0.5 mg/kg via a single intravenous infusion over a 30- to 45-minute period. Only a single trial of intranasal esketamine was identified. At 4 hours post-infusion, treatment with ketamine was associated with a significant reduction in suicidal ideation scores (standardised mean difference = -0.51, 95% confidence interval = [-1.00, -0.03]), which persisted until 72 hours post-infusion (time points between 12 and 24 hours: standardised mean difference = -0.63, 95% confidence interval = [-0.99, -0.26]; between 24 and 72 hours: standardised mean difference = -0.57, 95% confidence interval = [-0.99, -0.14]), but not thereafter. However, there was marked heterogeneity of results. In a single trial of esketamine, marginal effects on suicidal ideation were observed. In terms of actual suicidal behaviour, there were virtually no data on effects of ketamine or esketamine. CONCLUSION: A single infusion of ketamine may have a short-term (up to 72 hours) beneficial impact on suicidal thoughts. While confirmation of these results in further trials is needed, they suggest possible use of ketamine to treat acute suicidality. Means of sustaining any anti-suicidal effect need to be found.

Defining the remarkable structural malleability of a bacterial surface protein Rib domain implicated in infection.

Streptococcus groups A and B cause serious infections, including early onset sepsis and meningitis in newborns. Rib domain-containing surface proteins are found associated with invasive strains and elicit protective immunity in animal models. Yet, despite their apparent importance in infection, the structure of the Rib domain was previously unknown. Structures of single Rib domains of differing length reveal a rare case of domain atrophy through deletion of 2 core antiparallel strands, resulting in the loss of an entire sheet of the ß-sandwich from an immunoglobulin-like fold. Previously, observed variation in the number of Rib domains within these bacterial cell wall-attached proteins has been suggested as a mechanism of immune evasion. Here, the structure of tandem domains, combined with molecular dynamics simulations and small angle X-ray scattering, suggests that variability in Rib domain number would result in differential projection of an N-terminal host-colonization domain from the bacterial surface. The identification of 2 further structures where the typical B-D-E immunoglobulin ß-sheet is replaced with an α-helix further confirms the extensive structural malleability of the Rib domain.

Age, growth, mortality and reproductive seasonality of jolthead porgy, Calamus bajonado, from Florida waters.

Ages of jolthead porgy (Calamus bajonado Schneider 1801) (n = 635) from Florida commercial and recreational fisheries from 2008-2016 were determined using sectioned sagittal otoliths. We determined, using edge-type analysis, that opaque zones were annular, forming March-June (peaking in April). Jolthead porgy ranged from 1-13 years, and the largest fish measured 680 mm TL (total length, mm). Body size relationships for jolthead porgy were TL = 1.09FL + 20.44 (n = 622, r2 = 0.99), FL = 0.90 TL -14.26 (n = 622, r2 = 0.99), and W = 1.1 × 10-5 TL3.06 (n = 577), where W is total weight (grams, g) and FL is fork length (mm). The von Bertalanffy growth equation for jolthead porgy was Lt  = 737(1 - e-0.14(t+2.02)) (n = 635). Point estimate of natural mortality was M = 0.32, while age-specific estimates of M ranged from 0.58-0.17 y-1 for ages 1-13. Catch curve analysis estimated the instantaneous rate of total mortality Z = 0.70, while instantaneous rate of fishing mortality F was 0.38. Macroscopic staging of female gonads indicated the presence of hydrated oocytes from December-March, and GSI data indicates that peak spawning in females occurs during March. This study presents the first published findings of life history parameters for jolthead porgy from the Atlantic waters off the southeastern United States.

Investigating the Structural Compaction of Biomolecules Upon Transition to the Gas-Phase Using ESI-TWIMS-MS.

Collision cross-section (CCS) measurements obtained from ion mobility spectrometry-mass spectrometry (IMS-MS) analyses often provide useful information concerning a protein's size and shape and can be complemented by modeling procedures. However, there have been some concerns about the extent to which certain proteins maintain a native-like conformation during the gas-phase analysis, especially proteins with dynamic or extended regions. Here we have measured the CCSs of a range of biomolecules including non-globular proteins and RNAs of different sequence, size, and stability. Using traveling wave IMS-MS, we show that for the proteins studied, the measured CCS deviates significantly from predicted CCS values based upon currently available structures. The results presented indicate that these proteins collapse to different extents varying on their elongated structures upon transition into the gas-phase. Comparing two RNAs of similar mass but different solution structures, we show that these biomolecules may also be susceptible to gas-phase compaction. Together, the results suggest that caution is needed when predicting structural models based on CCS data for RNAs as well as proteins with non-globular folds. Graphical Abstract ᅟ.

Preliminary observations on the age and growth of dog snapper (Lutjanus jocu) and mahogany snapper (Lutjanus mahogoni) from the Southeastern U.S.

Dog snapper (Lutjanus jocu Bloch and Schneider 1801) and mahogany snapper (Lutjanus mahogoni Cuvier 1828) are infrequently caught snappers in the southeastern U.S. primarily occurring off of southern Florida. The species were opportunistically sampled from commercial and recreational fisheries in the southeastern U.S. from 1979 to 2015. Fish were aged (56 dog snapper and 54 mahogany snapper) by counting opaque zones on sectioned sagittal otoliths. Otoliths of both species were easily interpretable and agreement between readers was acceptable. Analysis of otolith edge-type revealed that annuli formed between May and July on both species. Dog snapper ranged from 200-837 mm total length (TL) and ages 2-33, while mahogany snapper ranged from 270-416 mm TL and ages 2-18. The Von Bertalanffy growth equations were Lt  = 746(1-e(-0.20(t-0.32))) and Lt  = 334(1-e(0.31(t+1.19))) for dog snapper and mahogany snapper, respectively. The weight-length relations were W = 1.31 × 10-5L3.03(n = 78, r2 = 0.99) and W = 5.40 × 10-6L3.15(n = 79, r2 = 0.79) for dog snapper and mahogany snapper, respectively, where W = whole weight in grams.

Smoking Behavior and Exposure: Results of a Menthol Cigarette Cross-over Study.

OBJECTIVE: Our objective was to improve understanding of the differences in use behavior and exposure when smoking menthol and non-menthol cigarettes using a 2-part cross-over design. METHODS: Adult daily smokers were assigned randomly to alternate between 2 weeks of exclusively smoking a menthol test cigarette or a non-menthol test cigarette. Urine and saliva were collected for biomarker measurements; carbon monoxide (CO) was measured, and participants smoked test cigarettes through a CreSS® smoking topography device during 3 clinic visits. Participants turned in their cigarette butts from the test periods for determination of mouth level nicotine and completed subjective questionnaires related to the test cigarettes. RESULTS: Regardless of cigarette preference, participants had higher salivary cotinine when smoking the non-menthol test cigarette, but there were no significant differences detected in urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol between the 2 test cigarettes. Mouth level nicotine, puff volume, and puff duration were significantly higher when smoking the menthol brand. Both menthol and non-menthol smokers reported significantly lower enjoyment and satisfaction scores for test cigarettes compared with their brand of choice. CONCLUSIONS: Our results suggest that mentholation has an effect on measures of smoking behavior and that mouth level nicotine is a useful indicator of between-brand smoke exposure.

Learning: Contemplating the unexamined core of Learning Health Systems.

INTRODUCTION: The current Learning Health Systems literature affords insufficient attention to the process of learning. In response, Billings Clinic focused on how to advance its learning capabilities and subsequently to contribute new insights into the process of learning to the LHS literature. METHODS: An environmental scan was conducted, including the grey literature (eg, technical reports and white papers) and peer-reviewed research publications. Semistructured interviews were also conducted with Clinic staff members to determine the motivation of their engagement in meaningful quality improvement, or learning, initiatives. RESULTS: Six learning principles emerged from the literature review and staff interviews: (1) draw on wisdom of groups and value connections; (2) embrace sensemaking over decision making in dealing with the unexpected; (3) bring diverse perspectives to complex challenges; (4) animate people, provide direction, update regularly, and interact respectfully; (5) appreciate the power and ubiquity of emergent change and the limitations of planned change; and (6) concentrate on small wins and characterize challenges as mere problems. Examples of how these principles are beginning to influence how learning and improvement are understood and approached at Billings Clinic are described and serve as illustrations of the principles in action. CONCLUSION: Becoming adept in learning is essential to realizing the vision of Learning Health Systems-to harness science, clinical research, and information generated by digital technology to inform and accelerate improvement in quality health care. This article seeks to contribute to greater understanding of this process by sharing a set of principles that are proving useful at one health care organization and to a more comprehensive conceptualization of Learning Health Systems.

Age, growth, and natural mortality of schoolmaster (Lutjanus apodus) from the southeastern United States.

Ages of schoolmaster (n = 136) from the southeastern Florida coast from 1981-2015 were determined using sectioned sagittal otoliths. Opaque zones were annular, forming March-July (peaking in May-June). Schoolmaster ranged in age from 1-42 years; the largest fish measured 505 mm total length (TL) and was 19 years old. The oldest fish measured 440 mm TL. Estimated body size relationships for schoolmaster were: W = 9.26 × 10-6TL3.11 (n = 256, r2 = 0.95); W = 2.13 × 10-5FL2.99 (n = 161, r2 = 0.95); TL = 1.03 FL + 10.36 (n = 143, r2 = 0.99); and FL = 0.96 TL - 8.41 (n = 143, r2 = 0.99), where W = whole weight in g, FL = fork length in mm, and TL in mm. The fitted von Bertalanffy growth equation was: Lt = 482 (1 - e-0.12(t+2.79)) (n = 136). Based on published life history relationships, a point estimate of natural mortality for schoolmaster was M = 0.10, while age-specific estimates of M ranged from 1.57-0.18 for ages 1-42.

Disorder drives cooperative folding in a multidomain protein.

Many human proteins contain intrinsically disordered regions, and disorder in these proteins can be fundamental to their function-for example, facilitating transient but specific binding, promoting allostery, or allowing efficient posttranslational modification. SasG, a multidomain protein implicated in host colonization and biofilm formation in Staphylococcus aureus, provides another example of how disorder can play an important role. Approximately one-half of the domains in the extracellular repetitive region of SasG are intrinsically unfolded in isolation, but these E domains fold in the context of their neighboring folded G5 domains. We have previously shown that the intrinsic disorder of the E domains mediates long-range cooperativity between nonneighboring G5 domains, allowing SasG to form a long, rod-like, mechanically strong structure. Here, we show that the disorder of the E domains coupled with the remarkable stability of the interdomain interface result in cooperative folding kinetics across long distances. Formation of a small structural nucleus at one end of the molecule results in rapid structure formation over a distance of 10 nm, which is likely to be important for the maintenance of the structural integrity of SasG. Moreover, if this normal folding nucleus is disrupted by mutation, the interdomain interface is sufficiently stable to drive the folding of adjacent E and G5 domains along a parallel folding pathway, thus maintaining cooperative folding.

Allosteric Regulation of Fibronectin/α5ß1 Interaction by Fibronectin-Binding MSCRAMMs.

Adherence of microbes to host tissues is a hallmark of infectious disease and is often mediated by a class of adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules). Numerous pathogens express MSCRAMMs that specifically bind the heterodimeric human glycoprotein fibronectin (Fn). In addition to roles in adhesion, Fn-binding MSCRAMMs exploit physiological Fn functions. For example, several pathogens can invade host cells by a mechanism whereby MSCRAMM-bound Fn bridges interaction with α5ß1 integrin. Here, we investigate two Fn-binding MSCRAMMs, FnBPA (Staphylococcus aureus) and BBK32 (Borrelia burgdorferi) to probe structure-activity relationships of MSCRAMM-induced Fn/α5ß1integrin activation. Circular dichroism, fluorescence resonance energy transfer, and dynamic light scattering techniques uncover a conformational rearrangement of Fn involving domains distant from the MSCRAMM binding site. Surface plasmon resonance experiments demonstrate a significant enhancement of Fn/α5ß1 integrin affinity in the presence of FnBPA or BBK32. Detailed kinetic analysis of these interactions reveal that this change in affinity can be attributed solely to an increase in the initial Fn/α5ß1 on-rate and that this rate-enhancement is dependent on high-affinity Fn-binding by MSCRAMMs. These data implicate MSCRAMM-induced perturbation of specific intramolecular contacts within the Fn heterodimer resulting in activation by exposing previously cryptic α5ß1 interaction motifs. By correlating structural changes in Fn to a direct measurement of increased Fn/α5ß1 affinity, this work significantly advances our understanding of the structural basis for the modulation of integrin function by Fn-binding MSCRAMMs.