G3BP1 Is a Tunable Switch that Triggers Phase Separation to Assemble Stress Granules.

The mechanisms underlying ribonucleoprotein (RNP) granule assembly, including the basis for establishing and maintaining RNP granules with distinct composition, are unknown. One prominent type of RNP granule is the stress granule (SG), a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to stress. Here, we show that SGs assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein-RNA interaction network. The central node of this network is G3BP1, which functions as a molecular switch that triggers RNA-dependent LLPS in response to a rise in intracellular free RNA concentrations. Moreover, we show that interplay between three distinct intrinsically disordered regions (IDRs) in G3BP1 regulates its intrinsic propensity for LLPS, and this is fine-tuned by phosphorylation within the IDRs. Further regulation of SG assembly arises through positive or negative cooperativity by extrinsic G3BP1-binding factors that strengthen or weaken, respectively, the core SG network.

Cancer Mutations of the Tumor Suppressor SPOP Disrupt the Formation of Active, Phase-Separated Compartments.

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

PaCO2 trajectories in mechanically ventilated patients with COVID-19: A population-based cohort study.

OBJECTIVE: To identify PaCO2 trajectories and assess their associations with mortality in critically ill patients with coronavirus disease 2019 (COVID-19) during the first and second waves of the pandemic in Denmark. DESIGN: A population-based cohort study with retrospective data collection. PATIENTS: All COVID-19 patients were treated in eight intensive care units (ICUs) in the Capital Region of Copenhagen, Denmark, between March 1, 2020 and March 31, 2021. MEASUREMENTS: Data from the electronic health records were extracted, and latent class analyses were computed based on up to the first 3 weeks of mechanical ventilation to depict trajectories of PaCO2 levels. Multivariable Cox regression analyses were used to calculate adjusted hazard ratios (aHRs) for Simplified Acute Physiology Score 3, sex and age with 95% confidence intervals (CIs) for death according to PaCO2 trajectories. MAIN RESULTS: In latent class trajectory models, including 25,318 PaCO2 measurements from 244 patients, three PaCO2 latent class trajectories were identified: a low isocapnic (Class I; n = 130), a high isocapnic (Class II; n = 80), as well as a progressively hypercapnic (Class III; n = 34) trajectory. Mortality was higher in Class II [aHR: 2.16 {1.26-3.68}] and Class III [aHR: 2.97 {1.63-5.40}]) compared to Class I (reference). CONCLUSION: Latent class analysis of arterial blood gases in mechanically ventilated COVID-19 patients identified distinct PaCO2 trajectories, which were independently associated with mortality.

Interplay of folded domains and the disordered low-complexity domain in mediating hnRNPA1 phase separation.

Liquid-liquid phase separation underlies the membrane-less compartmentalization of cells. Intrinsically disordered low-complexity domains (LCDs) often mediate phase separation, but how their phase behavior is modulated by folded domains is incompletely understood. Here, we interrogate the interplay between folded and disordered domains of the RNA-binding protein hnRNPA1. The LCD of hnRNPA1 is sufficient for mediating phase separation in vitro. However, we show that the folded RRM domains and a folded solubility-tag modify the phase behavior, even in the absence of RNA. Notably, the presence of the folded domains reverses the salt dependence of the driving force for phase separation relative to the LCD alone. Small-angle X-ray scattering experiments and coarse-grained MD simulations show that the LCD interacts transiently with the RRMs and/or the solubility-tag in a salt-sensitive manner, providing a mechanistic explanation for the observed salt-dependent phase separation. These data point to two effects from the folded domains: (i) electrostatically-mediated interactions that compact hnRNPA1 and contribute to phase separation and (ii) increased solubility at higher ionic strengths mediated by the folded domains. The interplay between disordered and folded domains can modify the dependence of phase behavior on solution conditions and can obscure signatures of physicochemical interactions underlying phase separation.

Combining systems thinking approaches and implementation science constructs within community-based prevention: a systematic review.

BACKGROUND: Systems science offers methods for designing population health interventions while implementation science provides specific guidance for successful implementation. Integrating systems and implementation science may strengthen implementation and enhance and sustain systemic change to achieve system-level outcomes. Little is known about the extent to which these two approaches have been integrated to date. This review aimed to identify and synthesise the peer-reviewed literature that has reported the combined use of systems thinking approaches and implementation science constructs (within the same study), to deliver population health interventions. METHODS: A systematic literature search of peer-reviewed original research was conducted across six databases from 2009 to 2021. Journal manuscripts were included if they: (1) reported on a population health study conducted in a community, (2) reported the use of a systems method in the design of the intervention, and (3) used an implementation science theory, framework or model in the delivery of the intervention. Data extracted related to the specific systems methods and definitions and implementation science constructs used. The Mixed Methods Appraisal Tool (MMAT) was used to assess study quality. RESULTS: Of the 9086 manuscripts returned, 320 manuscripts were included for full-text review. Of these, 17 manuscripts that reported on 14 studies were included in the final extraction. The most frequently reported systems methods were a 'whole of community systems approach' (n = 4/14) and 'community-based system dynamics' (n = 2/14). Nineteen different implementation science theories, frameworks and models were used for intervention delivery, with RE-AIM being the only framework used in more than one study. CONCLUSION: There are few published peer-reviewed studies using systems thinking and implementation science for designing and delivering population health interventions. An exploration of synergies is worthwhile to operationalise alignment and improve implementation of systems thinking approaches. Review protocol registration PROSPERO CRD42021250419.

Pacific Healthy Islands Vision: success factors and challenges faced by health promotion programs.

The World Health Organization's (WHO) Western Pacific Regional Office developed the biennial Healthy Islands Recognition Awards (HIA) in 2009 to reinforce the Healthy Islands vision and encourage countries to continue to innovate and demonstrate effective and efficient ways of promoting and protecting population health. This research aimed to identify characteristics of and challenges for successful health promotion in the Pacific. The research was undertaken to develop practical guidance for other groups in the Pacific Islands interested in supporting Healthy Islands. We used a qualitative case study to review 2013 and 2015 HIA awardees from eight Pacific Island countries and territories using a set of questions drawn from the HIA application criteria. In 2015-2016, 35 key informant interviews and a review of program documents were undertaken. This was followed by a workshop with representatives from three HIA awardees to further develop recommendations. We reviewed eight programs targeting healthy eating, physical activity, healthy settings and sanitation. Using evidence, careful planning, building capacity, developing partnerships, strengthening and reorientating networks, ensuring accountability and conducting evaluation were keys to the success of healthy islands projects. Considering the local setting and community was perhaps the most crucial theme amongst the programs examined. Challenges included funding and capacity constraints, maintaining commitment and prioritisation, maintaining communication and coordination and technical challenges. Success factors, challenges and recommendations aligned well with mainstream health promotion literature, although some important distinctions exist. Further research is needed to guide successful health promotion practice in the Pacific.

A framework for transforming the professional identity and brand image of All Nurses as Leaders.

BACKGROUND: The professional identity and brand image of nurses as leaders have not kept pace with the roles and scope of contemporary nursing practice. PURPOSE: To provide a framework to transform the professional identity and brand image of nursing from a caring discipline to one of leaders. METHODS: A Consensus Development Workgroup (CDW) design was used between the International Society for Professional Identity in Nursing (ISPIN) and the Institute for Brand Image of Nursing (IBIN) to advance the concept of All Nurses as Leaders across all settings and the public domain. DISCUSSION: The goal is to occupy a position in the minds of all stakeholders that differentiates nursing in a manner that is positive, relevant, accurate, desirable, and consistent over time. CONCLUSION: Current outcomes are endorsements, evidence-based strategies, and a framework to deconstruct the current brand image and align it with the desired brand image of All Nurses as Leaders.

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing.

TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.

Protein Network Structure Enables Switching between Liquid and Gel States.

Biomolecular condensates are emerging as an important organizational principle within living cells. These condensed states are formed by phase separation, yet little is known about how material properties are encoded within the constituent molecules and how the specificity for being in different phases is established. Here we use analytic theory to explain the phase behavior of the cancer-related protein SPOP and its substrate DAXX. Binary mixtures of these molecules have a phase diagram that contains dilute liquid, dense liquid, and gel states. We show that these discrete phases appear due to a competition between SPOP-DAXX and DAXX-DAXX interactions. The stronger SPOP-DAXX interactions dominate at sub-stoichiometric DAXX concentrations leading to the formation of cross-linked gels. The theory shows that the driving force for gel formation is not the binding energy, but rather the entropy of distributing DAXX molecules on the binding sites. At high DAXX concentrations the SPOP-DAXX interactions saturate, which leads to the dissolution of the gel and the appearance of a liquid phase driven by weaker DAXX-DAXX interactions. This competition between interactions allows multiple dense phases to form in a narrow region of parameter space. We propose that the molecular architecture of phase-separating proteins governs the internal structure of dense phases, their material properties and their functions. Analytical theory can reveal these properties on the long length and time scales relevant to biomolecular condensates.

Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1's ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

Competent with patients and populations: integrating public health into a medical program.

BACKGROUND: As the global burden of chronic disease grows, and infectious disease threats loom large, the need for medical graduates with expertise in public health medicine (PHM) is growing. A recurrent challenge is integrating this broad knowledge into crowded medical curricula and making PHM relevant. This study describes the process of integrating public health content into an Australian graduate entry medical course. METHODS: A redesign of the PHM curriculum at Deakin University School of Medicine was conducted in 2014 to make the curriculum practice-based and solution-oriented. Central to the redesign was the development of a curriculum map. RESULTS: Public health is now taught from a practice-based framework adapted from the World Health Organization emphasizing skills aligned with the Australasian Faculty of Public Health Medicine domains that prepare students for specialisation. Learning outcomes are structured to build depth and application in student knowledge. Mapping the curriculum provided the ability to measure alignment of learning outcomes with course, university and accrediting body outcomes. Regular feedback from students indicates engagement has improved along with perceived relevance to future careers. CONCLUSIONS: Doctors with public health skills are increasingly sought after in Australia, particularly in rural areas. Deakin graduates are well placed to meet this demand.

Relationship of Sequence and Phase Separation in Protein Low-Complexity Regions.

Liquid-liquid phase separation seems to play critical roles in the compartmentalization of cells through the formation of biomolecular condensates. Many proteins with low-complexity regions are found in these condensates, and they can undergo phase separation in vitro in response to changes in temperature, pH, and ion concentration. Low-complexity regions are thus likely important players in mediating compartmentalization in response to stress. However, how the phase behavior is encoded in their amino acid composition and patterning is only poorly understood. We discuss here that polymer physics provides a powerful framework for our understanding of the thermodynamics of mixing and demixing and for how the phase behavior is encoded in the primary sequence. We propose to classify low-complexity regions further into subcategories based on their sequence properties and phase behavior. Ongoing research promises to improve our ability to link the primary sequence of low-complexity regions to their phase behavior as well as the emerging miscibility and material properties of the resulting biomolecular condensates, providing mechanistic insight into this fundamental biological process across length scales.

Inflammatory cytokines down-regulate the barrier-protective prostasin-matriptase proteolytic cascade early in experimental colitis.

Compromised gastrointestinal barrier function is strongly associated with the progressive and destructive pathologies of the two main forms of irritable bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Matriptase is a membrane-anchored serine protease encoded by suppression of tumorigenicity-14 (ST14) gene, which is critical for epithelial barrier development and homeostasis. Matriptase barrier-protective activity is linked with the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin, which is a co-factor for matriptase zymogen activation. Here we show that mRNA and protein expression of both matriptase and prostasin are rapidly down-regulated in the initiating inflammatory phases of dextran sulfate sodium (DSS)-induced experimental colitis in mice, and, significantly, the loss of these proteases precedes the appearance of clinical symptoms, suggesting their loss may contribute to disease susceptibility. We used heterozygous St14 hypomorphic mice expressing a promoter-linked ß-gal reporter to show that inflammatory colitis suppresses the activity of the St14 gene promoter. Studies in colonic T84 cell monolayers revealed that barrier disruption by the colitis-associated Th2-type cytokines, IL-4 and IL-13, down-regulates matriptase as well as prostasin through phosphorylation of the transcriptional regulator STAT6 and that inhibition of STAT6 with suberoylanilide hydroxamic acid (SAHA) restores protease expression and reverses cytokine-induced barrier dysfunction. Both matriptase and prostasin are significantly down-regulated in colonic tissues from human subjects with active ulcerative colitis or Crohn's disease, implicating the loss of this barrier-protective protease pathway in the pathogenesis of irritable bowel disease.

Strategies to Recruit the Next Generation of Nursing Leadership Talent.

OBJECTIVE: The purpose of this study was to explore how generational differences contribute to challenges in recruiting the next generation of talent into nursing administration roles. BACKGROUND: The current workforce is aging. Nursing administrative roles, which for the purpose of this study are defined as nurse managers, are increasingly difficult to fill with leaders from upcoming Generations X and Y. METHODS: A descriptive, nonexperimental study was undertaken using a convenience sample. An electronic survey was distributed to explore the impact a set of interventions would have on recruiting into nursing administration roles. Then differences between generations were analyzed. RESULTS: Respondents indicated 45% of the interventions have a moderate to high impact on their consideration for a position in administration. Statistical significance between generations was identified on 4 interventions related to work-life harmony. CONCLUSIONS: Fiscally conservative interventions exist to assist hospital administrators with succession planning among nurse managers. When recruiting Generations X and Y candidates, interventions supporting work-life harmony should be emphasized.

Evaluating the effectiveness of risk minimisation measures: the application of a conceptual framework to Danish real-world dabigatran data.

PURPOSE: For both marketing authorization holders and regulatory authorities, evaluating the effectiveness of risk minimization measures is now an integral part of pharmacovigilance in the European Union. The overall aim of activities in this area is to assess the performance of risk minimization measures implemented in order to ensure a positive benefit-risk balance in patients treated with a medicinal product. METHODS: Following a review of the relevant literature, we developed a conceptual framework consisting of four domains (data, knowledge, behaviour and outcomes) intended for the evaluation of risk minimization measures put into practice in the Danish health-care system. For the implementation of the framework, four classes of monitoring variables can be named and defined: patient descriptors, performance-related indicators of knowledge, behaviour and outcomes. RESULTS: We reviewed the features of the framework when applied to historical, real-world data following the introduction of dabigatran in Denmark for the prophylactic treatment of patients with non-valvular atrial fibrillation. CONCLUSIONS: The application of the framework provided useful graphical displays and an opportunity for a statistical evaluation (interrupted time series analysis) of a regulatory intervention. © 2017 Commonwealth of Australia. Pharmacoepidemiology & Drug Safety © 2017 John Wiley & Sons, Ltd.

Guiding Principles for Creating Value and Meaning for the Next Generation of Nurse Leaders.

The wave of retirements is beginning to hit nursing, taking a toll on our cadre of nursing leaders. Ongoing healthcare reform is adding to the stress burden and exerting an effect on nurse leader turnover. The next generation of leaders will likely come from our millennial nurses, who have different attitudes toward work than previous generations. The AONE assembled a panel of experts to create guidelines to assist nurse executives in recruiting the next generation of talent into formal leadership roles.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface.

Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation.

Many cell signaling events are coordinated by intrinsically disordered protein regions (IDRs) that undergo multisite Serine/Threonine phosphorylation. The conformational properties of these IDRs prior to and following multisite phosphorylation are directly relevant to understanding their functions. Here, we present results from biophysical studies and molecular simulations that quantify the conformational properties of an 81-residue IDR from the S. cerevisiae transcription factor Ash1. We show that the unphosphorylated Ash1 IDR adopts coil-like conformations that are expanded and well-solvated. This result contradicts inferences regarding global compaction that are derived from heuristics based on amino acid compositions for IDRs with low proline contents. Upon phosphorylation at ten distinct sites, the global conformational properties of pAsh1 are indistinguishable from those of unphosphorylated Ash1. This insensitivity derives from compensatory changes to the pattern of local and long-range intrachain contacts. We show that the conformational properties of Ash1 and pAsh1 can be explained in terms of the linear sequence patterning of proline and charged residues vis-à-vis all other residues. The sequence features of the Ash1 IDR are shared by many other IDRs that undergo multisite phosphorylation. Accordingly, we propose that our findings might be generalizable to other IDRs involved in cell signaling.