ATOMDANCE: Kernel-based denoising and choreographic analysis for protein dynamic comparison.

Comparative methods in molecular evolution and structural biology rely heavily upon the site-wise analysis of DNA sequence and protein structure, both static forms of information. However, it is widely accepted that protein function results from nanoscale nonrandom machine-like motions induced by evolutionarily conserved molecular interactions. Comparisons of molecular dynamics (MD) simulations conducted between homologous sites representative of different functional or mutational states can potentially identify local effects on binding interaction and protein evolution. In addition, comparisons of different (i.e., nonhomologous) sites within MD simulations could be employed to identify functional shifts in local time-coordinated dynamics indicative of logic gating within proteins. However, comparative MD analysis is challenged by the large fraction of protein motion caused by random thermal noise in the surrounding solvent. Therefore, properly denoised MD comparisons could reveal functional sites involving these machine-like dynamics with good accuracy. Here, we introduce ATOMDANCE, a user-interfaced suite of comparative machine learning-based denoising tools designed for identifying functional sites and the patterns of coordinated motion they can create within MD simulations. ATOMDANCE-maxDemon4.0 employs Gaussian kernel functions to compute site-wise maximum mean discrepancy between learned features of motion, thereby assessing denoised differences in the nonrandom motions between functional or evolutionary states (e.g., ligand bound versus unbound, wild-type versus mutant). ATOMDANCE-maxDemon4.0 also employs maximum mean discrepancy to analyze potential random amino acid replacements allowing for a site-wise test of neutral versus nonneutral evolution on the divergence of dynamic function in protein homologs. Finally, ATOMDANCE-Choreograph2.0 employs mixed-model analysis of variance and graph network to detect regions where time-synchronized shifts in dynamics occur. Here, we demonstrate ATOMDANCE's utility for identifying key sites involved in dynamic responses during functional binding interactions involving DNA, small-molecule drugs, and virus-host recognition, as well as understanding shifts in global and local site coordination occurring during allosteric activation of a pathogenic protease.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are adept at sharing adaptive allelic variation, with commensal species repeatedly transferring resistance to their pathogenic relative Neisseria gonorrhoeae. However, resistance in commensals is infrequently characterized, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection as epistatic and additive interactions coerce lineages along divergent evolutionary trajectories. Alternatively, similar genetic content present across species due to shared ancestry may constrain existing adaptive solutions. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome-or the reservoir of alleles within the genus as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the ß-lactam penicillin. After 20 days of selection, commensals evolved resistance to penicillin and azithromycin in 11/16 and 12/16 cases, respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection and mtrRCDE, penA, and rpoB for penicillin selection, thus supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. Though drug-selected loci were limited, we do identify novel resistance-imparting mutations. Continuing to explore paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.IMPORTANCENeisseria gonorrhoeae is a global threat to public health due to its rapid acquisition of antibiotic resistance to all first-line treatments. Recent work has documented that alleles acquired from close commensal relatives have played a large role in the emergence of resistance to macrolides and beta-lactams within gonococcal populations. However, commensals have been relatively underexplored for the resistance genotypes they may harbor. This leaves a gap in our understanding of resistance that could be rapidly acquired by the gonococcus through a known highway of horizontal gene exchange. Here, we characterize resistance mechanisms that can emerge in commensal Neisseria populations via in vitro selection to multiple antimicrobials and begin to define the number of paths to resistance. This study, and other similar works, may ultimately aid both surveillance efforts and clinical diagnostic development by nominating novel and conserved resistance mechanisms that may be at risk of rapid dissemination to pathogen populations.

Rolling the evolutionary dice: Neisseria commensals as proxies for elucidating the underpinnings of antibiotic resistance mechanisms and evolution in human pathogens.

Species within the genus Neisseria are especially adept at sharing adaptive allelic variation across species' boundaries, with commensal species repeatedly transferring resistance to their pathogenic relative N. gonorrhoeae. However, resistance in commensal Neisseria is infrequently characterized at both the phenotypic and genotypic levels, limiting our ability to predict novel and potentially transferable resistance mechanisms that ultimately may become important clinically. Unique evolutionary starting places of each Neisseria species will have distinct genomic backgrounds, which may ultimately control the fate of evolving populations in response to selection, as epistatic and additive interactions may coerce lineages along divergent evolutionary trajectories. However alternatively, similar genetic content present across species due to shared ancestry may constrain the adaptive solutions that exist. Thus, identifying the paths to resistance across commensals may aid in characterizing the Neisseria resistome - or the reservoir of alleles within the genus, as well as its depth. Here, we use in vitro evolution of four commensal species to investigate the potential for and repeatability of resistance evolution to two antimicrobials, the macrolide azithromycin and the ß-lactam penicillin. After 20 days of selection, commensals evolved elevated minimum inhibitory concentrations (MICs) to penicillin and azithromycin in 11/16 and 12/16 cases respectively. Almost all cases of resistance emergence converged on mutations within ribosomal components or the mtrRCDE efflux pump for azithromycin-based selection, and mtrRCDE or penA for penicillin selection; thus, supporting constrained adaptive solutions despite divergent evolutionary starting points across the genus for these particular drugs. However, continuing to explore the paths to resistance across different experimental conditions and genomic backgrounds, which could shunt evolution down alternative evolutionary trajectories, will ultimately flesh out the full Neisseria resistome.

"I've still got a job to go back to": the importance of early vocational rehabilitation after stroke.

PURPOSE: Returning to work is an important goal after stroke, not only as a recovery indicator but also for facilitating independent living and improved social identity. The aim of this study was to explore the lived experiences of vocational rehabilitation and the return to work pathway after stroke. METHOD: Qualitative data were collected through semi-structured interviews with purposively selected participants who had participated in a vocational rehabilitation trial. All participants were employed at the time of their stroke and were community-living. Interviews were undertaken by occupational therapists and were transcribed verbatim before data were thematically analysed using a framework approach. RESULTS: Sixteen participants were interviewed, seven received specialist vocational rehabilitation and nine received usual clinical rehabilitation. Three major themes were identified which highlighted the importance of tailored vocational rehabilitation to address the challenges that arise when returning to the workplace. Stroke survivors perceived the most beneficial components of the specialist vocational rehabilitation intervention to be employer liaison support, fatigue management, and support for cognition and executive processing skills. CONCLUSIONS: Vocational rehabilitation was perceived to provide an opportunity to influence working after stroke, although areas of unmet need were highlighted. Findings provide direction for the development of future stroke-specific vocational rehabilitation programs.

Psychosocial (emotional) and cognitive (memory and planning) changes along with post-stroke fatigue were perceived to be the greatest barriers faced on returning to work and should be key areas for intervention within a vocational rehabilitation program.Vocational rehabilitation interventions should be tailored to each participant, incorporating elements perceived to be important to stroke survivors, including workplace liaison, employer communication and psychological support.Workplaces may benefit from access to information about the range of physical, cognitive and language deficits associated with stroke, and the potential impact these may have on work skills.

Updating the Standards of Professional Competence in the Field of Toxicology: The Second Generation of Best Practice.

The American Board of Toxicology (ABT), in consultation with ACT Credentialing & Career Services (ACT), performed a practice analysis study of general toxicology in 2020-21. This work follows up on an initial practice analysis commissioned by the ABT and conducted in 2014-2015, results of which were published in 2016. The purpose of the current, second-generation study was to update and validate the existing process-based delineation of practice of general toxicologists, including major domains of responsibility and tasks performed in practice. In addition, the study included the review, update, and validation of the knowledge areas required by toxicologists developed by subject-matter experts (SMEs) that have been used for ABT examination development initiatives. Consistent with best practices in the field of credentialing, ABT also contracted with ACT to conduct 2 follow-on activities: a study to evaluate the reliability of a reduced-length ABT examination and a standard setting study to establish a valid passing score for the updated examination. In addition to informing ongoing ABT certification examination and question writing activities, it is anticipated that the results of this practice analysis will be of value to those responsible for developing graduate and undergraduate toxicology curricula, creating continuing education content, and authoring textbooks covering the contemporary practice of toxicology.

Doctors' perspectives on adhering to advance care directives when making medical decisions for patients: an Australian interview study.

OBJECTIVE: Advance care planning (ACP) assists people to identify their goals, values and treatment preferences for future care. Ideally, preferences are documented in an advance care directive (ACD) and used by doctors to guide medical decision-making should the patient subsequently lose their decision-making capacity. However, studies demonstrate that ACDs are not always adhered to by doctors in clinical practice. We aim to describe the attitudes and perspectives of doctors regarding ACD adherence and the utility of ACDs in clinical practice. DESIGN: Face-to-face semistructured interviews were conducted using three case-based vignettes to explore doctors' decision-making and attitudes towards ACDs. Transcripts were analysed using a thematic analysis. SETTING: Doctors from a variety of medical specialties and with varying experience levels were recruited from a large tertiary hospital in Melbourne, Australia. PARTICIPANTS: A total of 21 doctors were interviewed, 48% female (10/21). Most (19/21) reported having experience using ACDs. RESULTS: Four themes were identified: aligning with patient preferences (avoiding unwanted care, prioritising autonomy and anticipating family opposition), advocating best interests (defining futile care, relying on clinical judgement, rejecting unreasonable decisions and disregarding legal consequences), establishing validity (doubting rigour of the decision-making process, questioning patients' ability to understand treatment decisions, distrusting outdated preferences and seeking confirmation) and translating written preferences into practice (contextualising patient preferences, applying subjective terminology and prioritising emergency medical treatment). CONCLUSIONS: ACDs provide doctors with opportunities to align patient preferences with treatment and uphold patient autonomy. However, doctors experience decisional conflict when attempting to adhere to ACDs in practice, especially when they believe that adhering to the ACD is not in the patients' best interests, or if they doubt the validity of the ACD. Future ACP programmes should consider approaches to improve the validity and applicability of ACDs. In addition, there is a need for ethical and legal education to support doctors' knowledge and confidence in ACP and enacting ACDs.

Biological fate of low-calorie sweeteners.

With continued efforts to find solutions to rising rates of obesity and diabetes, there is increased interest in the potential health benefits of the use of low- and no-calorie sweeteners (LNCSs). Concerns about safety often deter the use of LNCSs as a tool in helping control caloric intake, even though the safety of LNCS use has been affirmed by regulatory agencies worldwide. In many cases, an understanding of the biological fate of the different LNSCs can help health professionals to address safety concerns. The objectives of this review are to compare the similarities and differences in the chemistry, regulatory status, and biological fate (including absorption, distribution, metabolism, and excretion) of the commonly used LNCSs: acesulfame potassium, aspartame, saccharin, stevia leaf extract (steviol glycoside), and sucralose. Understanding the biological fate of the different LNCSs is helpful in evaluating whether reports of biological effects in animal studies or in humans are indicative of possible safety concerns. Illustrations of the usefulness of this information to address questions about LNCSs include discussion of systemic exposure to LNCSs, the use of sweetener combinations, and the potential for effects of LNCSs on the gut microflora.

Muscarinic receptor-activated signal transduction pathways involved in the neuritogenic effect of astrocytes in hippocampal neurons.

Astrocytes have been shown to release factors that affect various aspects of neuronal development. We have previously shown that the acetylcholine analog carbachol, by activating muscarinic M(3) receptors in rat astrocytes, increases their ability to promote neuritogenesis in hippocampal neurons. This effect was mediated by an increased expression and release by astrocytes of several permissive factors, a most relevant of which was fibronectin. In the present study we investigated the signal transduction pathways involved in these effects of carbachol in astrocytes. Results show that multiple pathways are involved in the effects of carbachol on astrocyte-mediated increases in fibronectin expression and neuritogenesis. These include the phospholipase D pathway, leading to sequential activation of protein kinase C (PKC) ζ, p70S6 kinase and nuclear factor-κB; the phosphoinositide-3 kinase pathway; and the PKC ε pathway leading to activation of mitogen activated protein kinase. These pathways were shown to mediate the effect of carbachol on neurite outgrowth as well as the increased expression of fibronectin, further substantiating the important role of the latter in astrocyte-mediated neuritogenesis. Interference with these signaling pathways would be expected to impair astrocyte-neurons communication leading to impaired neuronal development.

Ethanol inhibits neuritogenesis induced by astrocyte muscarinic receptors.

In utero alcohol exposure can lead to fetal alcohol spectrum disorders, characterized by cognitive and behavioral deficits. In vivo and in vitro studies have shown that ethanol alters neuronal development. We have recently shown that stimulation of M(3) muscarinic receptors in astrocytes increases the synthesis and release of fibronectin, laminin, and plasminogen activator inhibitor-1, causing neurite outgrowth in hippocampal neurons. As M(3) muscarinic receptor signaling in astroglial cells is strongly inhibited by ethanol, we hypothesized that ethanol may also inhibit neuritogenesis in hippocampal neurons induced by carbachol-stimulated astrocytes. In the present study, we report that the effect of carbachol-stimulated astrocytes on hippocampal neuron neurite outgrowth was inhibited in a concentration-dependent manner (25-100 mM) by ethanol. This effect was because of the inhibition of the release of fibronectin, laminin, and plasminogen activator inhibitor-1. Similar effects on neuritogenesis and on the release of astrocyte extracellular proteins were observed after the incubation of astrocytes with carbachol in the presence of 1-butanol, another short-chain alcohol, which like ethanol is a competitive substrate for phospholipase D, but not by tert-butanol, its analog that is not a substrate for this enzyme. This study identifies a potential novel mechanism involved in the developmental effects of ethanol mediated by the interaction of ethanol with cell signaling in astrocytes, leading to an impairment in neuron-astrocyte communication.

Shotgun proteomics implicates extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by gene ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. One hundred and thirty three secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

Modulation of neuritogenesis by astrocyte muscarinic receptors.

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.