Triage: A Global Perspective.

Triage is a process by which patients are assessed, classified, and sorted based on their presenting complaint and clinical urgency, providing assurance for timely access to emergency care. The goal is to get the right person to the right place, in the right amount of time, for the right reason, and within the context of resource availability. In many countries, a standardized triage system, underpinned through the use of guidelines, is used to provide clinicians with support and guidance. Triage is a globally adopted principle, and although triage guidelines are used in many countries, no single system has been internationally adopted. This paper discusses the importance of how triage process standardization improves patient care, resource management, and benchmarking at local, national, and international levels by applying 5 internationally recognized triage systems to fictional case studies. Evaluation of similarities and differences in severity scores, with a gap analysis, occurs.

A peroxiredoxin-P38 MAPK scaffold increases MAPK activity by MAP3K-independent mechanisms.

Peroxiredoxins (Prdxs) utilize reversibly oxidized cysteine residues to reduce peroxides and promote H2O2 signal transduction, including H2O2-induced activation of P38 MAPK. Prdxs form H2O2-induced disulfide complexes with many proteins, including multiple kinases involved in P38 MAPK signaling. Here, we show that a genetically encoded fusion between a Prdx and P38 MAPK is sufficient to hyperactivate the kinase in yeast and human cells by a mechanism that does not require the H2O2-sensing cysteine of the Prdx. We demonstrate that a P38-Prdx fusion protein compensates for loss of the yeast scaffold protein Mcs4 and MAP3K activity, driving yeast into mitosis. Based on our findings, we propose that the H2O2-induced formation of Prdx-MAPK disulfide complexes provides an alternative scaffold and signaling platform for MAPKK-MAPK signaling. The demonstration that formation of a complex with a Prdx is sufficient to modify the activity of a kinase has broad implications for peroxide-based signal transduction in eukaryotes.

3D printing-enabled uniform temperature distributions in microfluidic devices.

Many microfluidic processes rely heavily on precise temperature control. Though internally-contained heaters have been developed using traditional fabrication methods, they are limited in their ability to isothermally heat a precisely defined volume. Advances in 3D printing have led to high resolution printers capable of using bio-compatible materials and achieving geometry resolutions near 20 µm. 3D printing's ability to create arbitrary 3D structures with an arbitrary 3D orientation as opposed to traditional microfluidic fabrication methods enables new three-dimensional heater geometries to be created. As examples, we demonstrate three new 3D heater geometries: a non-planar serpentine channel, a tapered helical channel, and a diamond channel. These new geometries are shown through finite element simulation to isothermally heat microfluidic channels of cross section 200 µm × 200 µm with a 0.1 °C temperature difference along up to 91% of a 10 mm length, compared to designs from the literature that are only able to have that same temperature distance over several µms. Finally, a set of design rules to create isothermal regions in 3D based on the desired temperature, heater pitch, heater gradient, and radial space around a target volume are detailed.

Blocking Polyphosphate Mobilization Inhibits Pho4 Activation and Virulence in the Pathogen Candida albicans.

The ability of pathogenic fungi to obtain essential nutrients from the host is vital for virulence. In Candida albicans, acquisition of the macronutrient phosphate is regulated by the Pho4 transcription factor and is important for both virulence and resistance to host-encountered stresses. All cells store phosphate in the form of polyphosphate (polyP), a ubiquitous polymer comprising tens to hundreds of phosphate residues. Release of phosphate from polyP is one of the first responses evoked in response to phosphate starvation, and here, we sought to explore the importance of polyP mobilization in the pathobiology of C. albicans. We found that two polyphosphatases, Ppn1 and Ppx1, function redundantly to release phosphate from polyP in C. albicans. Strikingly, we reveal that blocking polyP mobilization prevents the activation of the Pho4 transcription factor: following Pi starvation, Pho4 fails to accumulate in the nucleus and induce Pi acquisition genes in ppn1Δ ppx1Δ cells. Consequently, ppn1Δ ppx1Δ cells display impaired resistance to the same range of stresses that require Pho4 for survival. In addition, cells lacking both polyphosphatases are exquisitely sensitive to DNA replication stress, indicating that polyP mobilization is needed to support the phosphate-demanding process of DNA replication. Blocking polyP mobilization also results in significant morphological defects, as ppn1Δ ppx1Δ cells form large pseudohypha-like cells that are resistant to serum-induced hypha formation. Thus, polyP mobilization impacts key processes important for the pathobiology of C. albicans, and consistent with this, we found that blocking this process attenuates the virulence of this important human fungal pathogen. IMPORTANCE Acquisition of the essential macronutrient phosphate is important for the virulence of Candida albicans, a major human fungal pathogen. All cells store phosphate as polyphosphate (polyP), which is rapidly mobilized when phosphate is limiting. Here, we identified the major phosphatases involved in releasing phosphate from polyP in C. albicans. By blocking this process, we found that polyP mobilization impacts many process that contribute to C. albicans pathogenesis. Notably, we found that blocking polyP mobilization inhibits activation of the Pho4 transcription factor, the master regulator of phosphate acquisition. In addition, cell cycle progression, stress resistance, morphogenetic switching, and virulence are all impaired in cells that cannot mobilize polyP. This study therefore provides new insight into the importance of polyP mobilization in promoting the virulence of C. albicans. As phosphate homeostasis strategies differ between fungal pathogen and host, this offers promise for the future development of antifungals.

Is it worth it? The costs and benefits of bringing a laptop to a university class.

Students often bring laptops to university classes, however, they do not limit their laptop use to class-related activity. Off-task laptop use occurs frequently in university classrooms and this use negatively impacts learning. The present study addresses whether potential benefits of class-related laptop use might mitigate the costs of off-task laptop activity. We used tracking software to monitor both class-related and off-task laptop use by undergraduate students enrolled in an introductory psychology course, and we observed how types of laptop use related to course performance. We found a positive correlation between class-related use and exam scores that was driven by viewing lecture slides during class. We also found a negative correlation between off-task laptop use and exam scores, but class-related activities did not predict an increase in off-task use. Thus, for students who constrain their laptop use to class-related activity, the benefits outweigh the costs. While a laptop may be beneficial for some, it is unclear which students are able to constrain themselves to class-related activities and whether the benefits of class-related laptop use obtained by slide viewing could be achieved by other means. Thus, students and educators should carefully consider the costs and benefits of laptop use in the classroom.

Planning for Chaos: Developing the Concept of Emergency Preparedness through the Experience of the Paramedic.

OBJECTIVE: Emergency preparedness is a developing specialty with a limited evidence base. Published literature primarily offers a retrospective view of experience, with few studies examining and understanding the individual lived experience of practitioners prospectively. This study explores paramedics' lived experience of emergency preparedness and applies that learning. METHODS: Thirteen paramedics were recruited through purposive sampling. Face-to-face semi-structured interviews explored their individual experiences of emergency preparedness, in line with the idiographic focus of Interpretative Phenomenological Analysis. RESULTS: Through data analysis, the following superordinate themes were identified for further discussion: self-determination, control, and experience-based practice. Participants appeared to value their role and the unpredictable environment in which they worked. Personal resilience, an area that they suggested is not covered effectively within individual preparation, was viewed as important. The participants articulated that risk, threat, uncertainty, safety, trust, and control were important concepts within individual preparedness. These paramedics valued practice-based knowledge and education as credible and transferrable to their clinical work. CONCLUSION: Evidence from this study suggests that standard emergency preparedness, with the focus at organizational level, is not sufficient for the individual workers or for an overall effective response. Dimensions of individual preparedness are presented, with the paramedic central to the experience within a conceptual model (the DiEP model), creating a new form of emergency preparedness that reflects the individual paramedic's experience.

Stress-Activated Protein Kinases in Human Fungal Pathogens.

The ability of fungal pathogens to survive hostile environments within the host depends on rapid and robust stress responses. Stress-activated protein kinase (SAPK) pathways are conserved MAPK signaling modules that promote stress adaptation in all eukaryotic cells, including pathogenic fungi. Activation of the SAPK occurs via the dual phosphorylation of conserved threonine and tyrosine residues within a TGY motif located in the catalytic domain. This induces the activation and nuclear accumulation of the kinase and the phosphorylation of diverse substrates, thus eliciting appropriate cellular responses. The Hog1 SAPK has been extensively characterized in the model yeast Saccharomyces cerevisiae. Here, we use this a platform from which to compare SAPK signaling mechanisms in three major fungal pathogens of humans, Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Despite the conservation of SAPK pathways within these pathogenic fungi, evidence is emerging that their role and regulation has significantly diverged. However, consistent with stress adaptation being a common virulence trait, SAPK pathways are important pathogenicity determinants in all these major human pathogens. Thus, the development of drugs which target fungal SAPKs has the exciting potential to generate broad-acting antifungal treatments.

Influence of ylHog1 MAPK kinase on Yarrowia lipolytica stress response and erythritol production.

Erythritol production is a unique response to hyperosmotic stress that is observed in a small group of yeasts, including Yarrowia lipolytica. This study investigated whether this unusual mechanism is regulated by the HOG pathway, well described in Saccharomyces cerevisiae. The gene YALI0E25135g was identified as the Y. lipolytica homologue of HOG1 and was found to be phosphorylated in response to hyperosmotic shock. Deletion of the gene caused a significant decrease in resistance to hyperosmotic stress and negatively affected erythritol production. Interestingly, the deletion strain yl-hog1Δ displayed significant morphological defects, with the cells growing in a filamentous form. Moreover, yl-hog1Δ cells were also resistant to the cell wall damaging agents Congo red and calcofluor white. Collectively, these results indicate that yl-Hog1 is crucial for the cellular response to hyperosmotic stress, plays a role in the induction of erythritol production, and potentially prevents cross-talk with different MAPK signalling pathways in the cell.

Hog1 Regulates Stress Tolerance and Virulence in the Emerging Fungal Pathogen Candida auris.

Candida auris has recently emerged as an important, multidrug-resistant fungal pathogen of humans. Comparative studies indicate that despite high levels of genetic divergence, C. auris is as virulent as the most pathogenic member of the genus, Candida albicans However, key virulence attributes of C. albicans, such as morphogenetic switching, are not utilized by C. auris, indicating that this emerging pathogen employs alternative strategies to infect and colonize the host. An important trait required for the pathogenicity of many fungal pathogens is the ability to adapt to host-imposed stresses encountered during infection. Here, we investigated the relative resistance of C. auris and other pathogenic Candida species to physiologically relevant stresses and explored the role of the evolutionarily conserved Hog1 stress-activated protein kinase (SAPK) in promoting stress resistance and virulence. In comparison to C. albicans, C. auris is relatively resistant to hydrogen peroxide, cationic stress, and cell-wall-damaging agents. However, in contrast to other Candida species examined, C. auris was unable to grow in an anaerobic environment and was acutely sensitive to organic oxidative-stress-inducing agents. An analysis of C. aurishog1Δ cells revealed multiple roles for this SAPK in stress resistance, cell morphology, aggregation, and virulence. These data demonstrate that C. auris has a unique stress resistance profile compared to those of other pathogenic Candida species and that the Hog1 SAPK has pleiotropic roles that promote the virulence of this emerging pathogen.IMPORTANCE The rapid global emergence and resistance of Candidaauris to current antifungal drugs highlight the importance of understanding the virulence traits exploited by this human fungal pathogen to cause disease. Here, we characterize the stress resistance profile of C. auris and the role of the Hog1 stress-activated protein kinase (SAPK) in stress resistance and virulence. Our findings that C. auris is acutely sensitive to certain stresses may facilitate control measures to prevent persistent colonization in hospital settings. Furthermore, our observation that the Hog1 SAPK promotes C. auris virulence akin to that reported for many other pathogenic fungi indicates that antifungals targeting Hog1 signaling would be broad acting and effective, even on emerging drug-resistant pathogens.

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs.

In all eukaryotic kingdoms, mitogen-activated protein kinases (MAPKs) play critical roles in cellular responses to environmental cues. These MAPKs are activated by phosphorylation at highly conserved threonine and tyrosine residues in response to specific inputs, leading to their accumulation in the nucleus and the activation of their downstream targets. A specific MAP kinase can regulate different downstream targets depending on the nature of the input signal, thereby raising a key question: what defines the stress-specific outputs of MAP kinases? We find that the Hog1 MAPK contributes to nitrosative-stress resistance in Candida albicans even though it displays minimal stress-induced phosphorylation under these conditions. We show that Hog1 becomes oxidized in response to nitrosative stress, accumulates in the nucleus, and regulates the nitrosative stress-induced transcriptome. Mutation of specific cysteine residues revealed that C156 and C161 function together to promote stress resistance, Hog1-mediated nitrosative-stress-induced gene expression, resistance to phagocytic killing, and C. albicans virulence. We propose that the oxidation of Hog1, rather than its phosphorylation, contributes to the nitrosative-stress-specific responses of this MAP kinase.IMPORTANCE Mitogen-activated protein kinases play key roles in the responses of eukaryotic cells to extracellular signals and are critical for environmental-stress resistance. The widely accepted paradigm is that MAP kinases are activated by phosphorylation, which then triggers their nuclear accumulation and the activation of target proteins and genes that promote cellular adaptation. Our data suggest that alternative forms of posttranslational modification can modulate MAP kinase functionality in Candida albicans We demonstrate that Hog1 is not significantly phosphorylated in response to nitrosative stress, yet it displays nuclear accumulation and contributes to the global transcriptional response to this stress, as well as promoting nitrosative-stress resistance. Instead, nitrosative stress triggers changes in the redox status of Hog1. We also show that specific Hog1 cysteine residues influence its activation of stress genes. Therefore, alternative posttranslational modifications appear to regulate the stress-specific outputs of MAP kinases.

Forward view: advancing health library and knowledge services in England.

This article is the fourth in a series on New Directions. The National Health Service is under pressure, challenged to meet the needs of an ageing population, whilst striving to improve standards and ensure decision making is underpinned by evidence. Health Education England is steering a new course for NHS library and knowledge services in England to ensure access to knowledge and evidence for all decision makers. Knowledge for Healthcare calls for service transformation, role redesign, greater coordination and collaboration. To meet user expectations, health libraries must achieve sustainable, affordable access to digital content. Traditional tasks will progressively become mechanised. Alongside supporting learners, NHS librarians and knowledge specialists will take a greater role as knowledge brokers, delivering business critical services. They will support the NHS workforce to signpost patients and the public to high-quality information. There is a need for greater efficiency and effectiveness through greater co-operation and service mergers. Evaluation of service quality will focus more on outcomes, less on counting. These changes require an agile workforce, fit for the future. There is a bright future in which librarians' expertise is used to mobilise evidence, manage and share knowledge, support patients, carers and families, optimise technology and social media and provide a keystone for improved patient care and safety.

Stress-induced nuclear accumulation is dispensable for Hog1-dependent gene expression and virulence in a fungal pathogen.

Stress-activated protein kinase (SAPK) pathways are evolutionarily conserved eukaryotic signalling modules that are essential for the virulence of human pathogenic fungi. The Hog1 SAPK in Candida albicans is robustly phosphorylated in response to a number of host-imposed stresses, and is essential for virulence. The current dogma is that stress-induced phosphorylation activates the SAPK, and promotes its nuclear accumulation that is necessary for the expression of SAPK-dependent stress-protective genes. Here we challenge this dogma. C. albicans strains were constructed in which Hog1 was either tethered to the plasma membrane or constitutively nuclear. Strikingly, tethering Hog1 to the plasma membrane did not abrogate stress resistance or stress-induced gene expression. Furthermore, preventing the nuclear accumulation of Hog1 had no impact on C. albicans virulence in two distinct models of systemic infection. However, tethering Hog1 to the plasma membrane did impact on signal fidelity, and on the magnitude and kinetics of the stress-induced phosphorylation of this SAPK. Taken together, these findings challenge the dogma that nuclear accumulation of SAPKs is a pre-requisite for SAPK-dependent gene expression, and reveal that stress-induced nuclear accumulation of Hog1 is dispensable for the virulence of a major human fungal pathogen.

Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation.

The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the 'p38-related' Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

Increasing extracellular H2O2 produces a bi-phasic response in intracellular H2O2, with peroxiredoxin hyperoxidation only triggered once the cellular H2O2-buffering capacity is overwhelmed.

Reactive oxygen species, such as H2O2, can damage cells but also promote fundamental processes, including growth, differentiation and migration. The mechanisms allowing cells to differentially respond to toxic or signaling H2O2 levels are poorly defined. Here we reveal that increasing external H2O2 produces a bi-phasic response in intracellular H2O2. Peroxiredoxins (Prx) are abundant peroxidases which protect against genome instability, ageing and cancer. We have developed a dynamic model simulating in vivo changes in Prx oxidation. Remarkably, we show that the thioredoxin peroxidase activity of Prx does not provide any significant protection against external rises in H2O2. Instead, our model and experimental data are consistent with low levels of extracellular H2O2 being efficiently buffered by other thioredoxin-dependent activities, including H2O2-reactive cysteines in the thiol-proteome. We show that when extracellular H2O2 levels overwhelm this buffering capacity, the consequent rise in intracellular H2O2 triggers hyperoxidation of Prx to thioredoxin-resistant, peroxidase-inactive form/s. Accordingly, Prx hyperoxidation signals that H2O2 defenses are breached, diverting thioredoxin to repair damage.

The GH130 Family of Mannoside Phosphorylases Contains Glycoside Hydrolases That Target ß-1,2-Mannosidic Linkages in Candida Mannan.

The depolymerization of complex glycans is an important biological process that is of considerable interest to environmentally relevant industries. ß-Mannose is a major component of plant structural polysaccharides and eukaryotic N-glycans. These linkages are primarily cleaved by glycoside hydrolases, although recently, a family of glycoside phosphorylases, GH130, have also been shown to target ß-1,2- and ß-1,4-mannosidic linkages. In these phosphorylases, bond cleavage was mediated by a single displacement reaction in which phosphate functions as the catalytic nucleophile. A cohort of GH130 enzymes, however, lack the conserved basic residues that bind the phosphate nucleophile, and it was proposed that these enzymes function as glycoside hydrolases. Here we show that two Bacteroides enzymes, BT3780 and BACOVA_03624, which lack the phosphate binding residues, are indeed ß-mannosidases that hydrolyze ß-1,2-mannosidic linkages through an inverting mechanism. Because the genes encoding these enzymes are located in genetic loci that orchestrate the depolymerization of yeast α-mannans, it is likely that the two enzymes target the ß-1,2-mannose residues that cap the glycan produced by Candida albicans. The crystal structure of BT3780 in complex with mannose bound in the -1 and +1 subsites showed that a pair of glutamates, Glu(227) and Glu(268), hydrogen bond to O1 of α-mannose, and either of these residues may function as the catalytic base. The candidate catalytic acid and the other residues that interact with the active site mannose are conserved in both GH130 mannoside phosphorylases and ß-1,2-mannosidases. Functional phylogeny identified a conserved lysine, Lys(199) in BT3780, as a key specificity determinant for ß-1,2-mannosidic linkages.

Glycan complexity dictates microbial resource allocation in the large intestine.

The structure of the human gut microbiota is controlled primarily through the degradation of complex dietary carbohydrates, but the extent to which carbohydrate breakdown products are shared between members of the microbiota is unclear. We show here, using xylan as a model, that sharing the breakdown products of complex carbohydrates by key members of the microbiota, such as Bacteroides ovatus, is dependent on the complexity of the target glycan. Characterization of the extensive xylan degrading apparatus expressed by B. ovatus reveals that the breakdown of the polysaccharide by the human gut microbiota is significantly more complex than previous models suggested, which were based on the deconstruction of xylans containing limited monosaccharide side chains. Our report presents a highly complex and dynamic xylan degrading apparatus that is fine-tuned to recognize the different forms of the polysaccharide presented to the human gut microbiota.