A Novel Comprehensive Algorithm for Evaluation of PICU Patients With New Fever or Instability.

OBJECTIVES: There is variation in microbiology testing among PICU patients with fever offering opportunities to reduce avoidable testing and treatment. Our objective is to describe the development and assess the impact of a novel comprehensive testing algorithm to support judicious testing practices and expanded diagnostic differentials for PICU patients with new fever or instability. DESIGN: A mixed-methods quality improvement study. SETTING: Single-center academic PICU and pediatric cardiac ICU. SUBJECTS: Admitted PICU patients and physicians. INTERVENTIONS: A multidisciplinary team developed a clinical decision-support algorithm. MEASUREMENTS AND MAIN RESULTS: We evaluated blood, endotracheal, and urine cultures, urinalyses, and broad-spectrum antibiotic use per 1,000 ICU patient-days using statistical process control charts and incident rate ratios (IRRs) and assessed clinical outcomes 24 months pre- and 18 months postimplementation. We surveyed physicians weekly for 12 months postimplementation. Blood cultures declined by 17% (IRR, 0.83; 95% CI, 0.77-0.89), endotracheal cultures by 26% (IRR, 0.74; 95% CI, 0.63-0.86), and urine cultures by 36% (IRR, 0.64; 95% CI, 0.56-0.73). There was an anticipated rise in urinalysis testing by 23% (IRR, 1.23; 95% CI, 1.14-1.33). Despite higher acuity and fewer brief hospitalizations, mortality, hospital, and PICU readmissions were stable, and PICU length of stay declined. Of the 108 physician surveys, 46 replied (43%), and 39 (85%) recently used the algorithm; 0 reported patient safety concerns, two (4%) provided constructive feedback, and 28 (61%) reported the algorithm improved patient care. CONCLUSIONS: A comprehensive fever algorithm was associated with reductions in blood, endotracheal, and urine cultures and anticipated increase in urinalyses. We detected no patient harm, and physicians reported improved patient care.

Biology of Stress Responses in Aging.

On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.

Males induce premature demise of the opposite sex by multifaceted strategies.

Interactions between the sexes negatively impact health in many species. In Caenorhabditis, males shorten the lifespan of the opposite sex-hermaphrodites or females. Here we use transcriptomic profiling and targeted screens to systematically uncover conserved genes involved in male-induced demise in C. elegans. Some genes (for example, delm-2, acbp-3), when knocked down, are specifically protective against male-induced demise. Others (for example, sri-40), when knocked down, extend lifespan with and without males, suggesting general mechanisms of protection. In contrast, many classical long-lived mutants are impacted more negatively than wild type by the presence of males, highlighting the importance of sexual environment for longevity. Interestingly, genes induced by males are triggered by specific male components (seminal fluid, sperm and pheromone), and manipulating these genes in combination in hermaphrodites induces stronger protection. One of these genes, the conserved ion channel delm-2, acts in the nervous system and intestine to regulate lipid metabolism. Our analysis reveals striking differences in longevity in single sex versus mixed sex environments and uncovers elaborate strategies elicited by sexual interactions that could extend to other species.

Culture Ordering for Patients with New-onset Fever: A Survey of Pediatric Intensive Care Unit Clinician Practices.

INTRODUCTION: Accurate assessment of infection in critically ill patients is vital to their care. Both indiscretion and under-utilization of diagnostic microbiology testing can contribute to inappropriate antibiotic administration or delays in diagnosis. However, indiscretion in diagnostic microbiology cultures may also lead to unnecessary tests that, if false-positive, would incur additional costs and unhelpful evaluations. This quality improvement project objective was to assess pediatric intensive care unit (PICU) clinicians' attitudes and practices around the microbiology work-up for patients with new-onset fever. METHODS: We developed and conducted a self-administered electronic survey of PICU clinicians at a single institution. The survey included 7 common clinical vignettes of PICU patients with new-onset fever and asked participants whether they would obtain central line blood cultures, peripheral blood cultures, respiratory aspirate cultures, cerebrospinal fluid cultures, urine cultures, and/or urinalyses. RESULTS: Forty-seven of 54 clinicians (87%) completed the survey. Diagnostic specimen ordering practices were notably heterogeneous. Respondents unanimously favored a decision-support algorithm to guide culture specimen ordering practices for PICU patients with fever (100%, N = 47). A majority (91.5%, N = 43) indicated that a decision-support algorithm would be a means to align PICU and consulting care teams when ordering culture specimens for patients with fever. CONCLUSION: This survey revealed variability of diagnostic specimen ordering practices for patients with new fever, supporting an opportunity to standardize practices. Clinicians favored a decision-support tool and thought that it would help align patient management between clinical team members. The results will be used to inform future diagnostic stewardship efforts.

Coordination of Substrate Binding and Protonation in the N. gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N. gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N. gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

Program evaluation of trauma-informed yoga for vulnerable populations.

BACKGROUND: Trauma is highly prevalent among vulnerable populations, including those who are incarcerated, in treatment for substance use, or seeking mental health services. Trauma-informed yoga seeks to create a safer yoga practice for individuals with a trauma history and may improve emotional and physical wellbeing. Thus, we conducted an evaluation of a trauma-informed yoga program to gain insight into participant experiences. METHODS: Trauma-informed yoga classes were led by trained volunteers and held in three sectors that work with vulnerable populations: corrections and reentry, substance use treatment and recovery, and community and mental health. Data were collected via anonymous survey using a retrospective pre-post design. The survey instrument captured reasons for student participation and perceived effects of yoga on emotional and physical wellbeing. RESULTS: Students were motivated to participate in yoga classes by expectations of physical, mental, and spiritual benefit. Students reported perceived improvements in emotional and physical wellbeing and greater use of self-regulation skills after starting yoga. CONCLUSION: Our findings suggest trauma-informed yoga is perceived as beneficial by vulnerable individuals, especially those in the correctional system or recovering from substance use. Our results support the value of offering trauma-informed yoga in institutionalized and community settings. Improvements in emotional and physical wellbeing warrant formal study.

Multidrug Resistance in Neisseria gonorrhoeae: Identification of Functionally Important Residues in the MtrD Efflux Protein.

A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

Insulin-like peptides and the mTOR-TFEB pathway protect Caenorhabditis elegans hermaphrodites from mating-induced death.

Lifespan is shortened by mating, but these deleterious effects must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death is caused by the loss of protection upon self-sperm depletion. Self-sperm maintains the expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of intestinal HLH-30/TFEB, a key pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death until self-sperm are exhausted, increasing the chances that mothers will survive through reproduction. Mothers combat males' hijacking of their IIS pathway by expressing an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize.

Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes.

Sexual interactions have a potent influence on health in several species, including mammals. Previous work in C. elegans identified strategies used by males to accelerate the demise of the opposite sex (hermaphrodites). But whether hermaphrodites evolved counter-strategies against males remains unknown. Here we discover that young C. elegans hermaphrodites are remarkably resistant to brief sexual encounters with males, whereas older hermaphrodites succumb prematurely. Surprisingly, it is not their youthfulness that protects young hermaphrodites, but the fact that they have self-sperm. The beneficial effect of self-sperm is mediated by a sperm-sensing pathway acting on the soma rather than by fertilization. Activation of this pathway in females triggers protection from the negative impact of males. Interestingly, the role of self-sperm in protecting against the detrimental effects of males evolved independently in hermaphroditic nematodes. Endogenous strategies to delay the negative effect of mating may represent a key evolutionary innovation to maximize reproductive success.

AlloMAPS: allosteric mutation analysis and polymorphism of signaling database.

AlloMAPS database provides data on the causality and energetics of allosteric communication obtained with the structure-based statistical mechanical model of allostery (SBSMMA). The database contains data on allosteric signaling in three sets of proteins and protein chains: (i) 46 proteins with comprehensively annotated functional and allosteric sites; (ii) 1908 protein chains from PDBselect set of chains with low (<25%) sequence identity; (iii) 33 proteins with more than 50 known pathological SNPs in each molecule. In addition to energetics of allosteric signaling between known functional and regulatory sites, allosteric modulation caused by the binding to these sites, by SNPs, and by mutations designated by the user can be explored. Allosteric Signaling Maps (ASMs), which are produced via the exhaustive computational scanning for stabilizing and destabilizing mutations and for the modulation range caused by the sequence position are available for each protein/protein chain in the database. We propose to use this database for evaluating the effects of allosteric signaling in the search for latent regulatory sites and in the design of allosteric sites and effectors. The database is freely available at: http://allomaps.bii.a-star.edu.sg.

The Aging Epigenome.

During aging, the mechanisms that normally maintain health and stress resistance strikingly decline, resulting in decrepitude, frailty, and ultimately death. Exactly when and how this decline occurs is unknown. Changes in transcriptional networks and chromatin state lie at the heart of age-dependent decline. These epigenomic changes are not only observed during aging but also profoundly affect cellular function and stress resistance, thereby contributing to the progression of aging. We propose that the dysregulation of transcriptional and chromatin networks is a crucial component of aging. Understanding age-dependent epigenomic changes will yield key insights into how aging begins and progresses and should lead to the development of new therapeutics that delay or even reverse aging and age-related diseases.

Shockingly Early: Chromatin-Mediated Loss of the Heat Shock Response.

In this issue of Molecular Cell, Labbadia and Morimoto (2015) show that there is a precipitous decline in stress resistance at the onset of reproduction in C. elegans and that this transition is regulated by changes in repressive chromatin marks.

Intersecting transcription networks constrain gene regulatory evolution.

Epistasis-the non-additive interactions between different genetic loci-constrains evolutionary pathways, blocking some and permitting others. For biological networks such as transcription circuits, the nature of these constraints and their consequences are largely unknown. Here we describe the evolutionary pathways of a transcription network that controls the response to mating pheromone in yeast. A component of this network, the transcription regulator Ste12, has evolved two different modes of binding to a set of its target genes. In one group of species, Ste12 binds to specific DNA binding sites, while in another lineage it occupies DNA indirectly, relying on a second transcription regulator to recognize DNA. We show, through the construction of various possible evolutionary intermediates, that evolution of the direct mode of DNA binding was not directly accessible to the ancestor. Instead, it was contingent on a lineage-specific change to an overlapping transcription network with a different function, the specification of cell type. These results show that analysing and predicting the evolution of cis-regulatory regions requires an understanding of their positions in overlapping networks, as this placement constrains the available evolutionary pathways.

Males shorten the life span of C. elegans hermaphrodites via secreted compounds.

How an individual's longevity is affected by the opposite sex is still largely unclear. In the nematode Caenorhabditis elegans, the presence of males accelerated aging and shortened the life span of individuals of the opposite sex (hermaphrodites), including long-lived or sterile hermaphrodites. The male-induced demise could occur without mating and required only exposure of hermaphrodites to medium in which males were once present. Such communication through pheromones or other diffusible substances points to a nonindividual autonomous mode of aging regulation. The male-induced demise also occurred in other species of nematodes, suggesting an evolutionary conserved process whereby males may induce the disposal of the opposite sex to save resources for the next generation or to prevent competition from other males.

Protein modularity, cooperative binding, and hybrid regulatory states underlie transcriptional network diversification.

We examine how different transcriptional network structures can evolve from an ancestral network. By characterizing how the ancestral mode of gene regulation for genes specific to a-type cells in yeast species evolved from an activating paradigm to a repressing one, we show that regulatory protein modularity, conversion of one cis-regulatory sequence to another, distribution of binding energy among protein-protein and protein-DNA interactions, and exploitation of ancestral network features all contribute to the evolution of a novel regulatory mode. The formation of this derived mode of regulation did not disrupt the ancestral mode and thereby created a hybrid regulatory state where both means of transcription regulation (ancestral and derived) contribute to the conserved expression pattern of the network. Finally, we show how this hybrid regulatory state has resolved in different ways in different lineages to generate the diversity of regulatory network structures observed in modern species.

Intercalation of a new tier of transcription regulation into an ancient circuit.

Changes in gene regulatory networks are a major source of evolutionary novelty. Here we describe a specific type of network rewiring event, one that intercalates a new level of transcriptional control into an ancient circuit. We deduce that, over evolutionary time, the direct ancestral connections between a regulator and its target genes were broken and replaced by indirect connections, preserving the overall logic of the ancestral circuit but producing a new behaviour. The example was uncovered through a series of experiments in three ascomycete yeasts: the bakers' yeast Saccharomyces cerevisiae, the dairy yeast Kluyveromyces lactis and the human pathogen Candida albicans. All three species have three cell types: two mating-competent cell forms (a and α) and the product of their mating (a/α), which is mating-incompetent. In the ancestral mating circuit, two homeodomain proteins, Mata1 and Matα2, form a heterodimer that directly represses four genes that are expressed only in a and α cells and are required for mating. In a relatively recent ancestor of K. lactis, a reorganization occurred. The Mata1-Matα2 heterodimer represses the same four genes (known as the core haploid-specific genes) but now does so indirectly through an intermediate regulatory protein, Rme1. The overall logic of the ancestral circuit is preserved (haploid-specific genes ON in a and α cells and OFF in a/α cells), but a new phenotype was produced by the rewiring: unlike S. cerevisiae and C. albicans, K. lactis integrates nutritional signals, by means of Rme1, into the decision of whether or not to mate.

Arginine-rich motifs present multiple interfaces for specific binding by RNA.

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.