Mapping N- to C-terminal allosteric coupling through disruption of a putative CD74 activation site in D-dopachrome tautomerase.

The macrophage migration inhibitory factor (MIF) protein family consists of MIF and D-dopachrome tautomerase (also known as MIF-2). These homologs share 34% sequence identity while maintaining nearly indistinguishable tertiary and quaternary structure, which is likely a major contributor to their overlapping functions, including the binding and activation of the cluster of differentiation 74 (CD74) receptor to mediate inflammation. Previously, we investigated a novel allosteric site, Tyr99, that modulated N-terminal catalytic activity in MIF through a "pathway" of dynamically coupled residues. In a comparative study, we revealed an analogous allosteric pathway in MIF-2 despite its unique primary sequence. Disruptions of the MIF and MIF-2 N termini also diminished CD74 activation at the C terminus, though the receptor activation site is not fully defined in MIF-2. In this study, we use site-directed mutagenesis, NMR spectroscopy, molecular simulations, in vitro and in vivo biochemistry to explore the putative CD74 activation region of MIF-2 based on homology to MIF. We also confirm its reciprocal structural coupling to the MIF-2 allosteric site and N-terminal enzymatic site. Thus, we provide further insight into the CD74 activation site of MIF-2 and its allosteric coupling for immunoregulation.

Performance of the European Society of Cardiology 0/1-hour algorithm with high-sensitivity cardiac troponin T at 90 days among patients with known coronary artery disease.

BACKGROUND: The European Society of Cardiology (ESC) 0/1-h high sensitivity troponin T (hs-cTnT) algorithm does not differentiate risk based on known coronary artery disease (CAD: prior myocardial infarction [MI], coronary revascularization, or ≥ 70% coronary stenosis). We recently evaluated its performance among patients with known CAD at 30-days, but little is known about its longer-term risk prediction. The objective of this study is to determine and compare the performance of the algorithm at 90-days among patients with and without known CAD. METHODS: We performed a pre-planned subgroup analysis of the STOP-CP cohort, which prospectively enrolled ED patients ≥21 years old with symptoms suggestive of ACS without ST-elevation on initial ECG across 8 US sites (1/25/2017-9/6/2018). Participants with 0- and 1-h hs-cTnT measures (Roche, Basel, Switzerland) were stratified into rule-out, observe, and rule-in groups using the ESC 0/1-h algorithm. Algorithm performance was tested among patients with or without known CAD, as determined by the treating provider. The primary outcome was cardiac death or MI at 90-days. Fisher's exact tests were used to compare 90-day event and rule-out rates between patients with and without known CAD. Negative predictive values (NPVs) for 90-day cardiac death or MI with exact 95% confidence intervals were calculated and compared using Fisher's exact test. RESULTS: The STOP-CP study accrued 1430 patients, of which 31.4% (449/1430) had known CAD. Cardiac death or MI at 90 days was more common in patients with known CAD than in those without [21.2% (95/449) vs. 10.0% (98/981); p < 0.001]. Using the ESC 0/1-h algorithm, 39.6% (178/449) of patients with known CAD and 66.1% (648/981) of patients without known CAD were ruled-out (p < 0.001). Among rule-out patients, 90-day cardiac death or MI occurred in 3.4% (6/178) of patients with known CAD and 1.2% (8/648) without known CAD (p = 0.09). NPV for 90-day cardiac death or MI was 96.6% (95%CI 92.8-98.8) among patients with known CAD and 98.8% (95%CI 97.6-99.5) in patients without known CAD (p = 0.09). CONCLUSION: Patients with known CAD who were ruled-out using the ESC 0/1-h hs-cTnT algorithm had a high rate of missed 90-day cardiac events, suggesting that the ESC 0/1-h hs-cTnT algorithm may not be safe for use among patients with known CAD. TRIAL REGISTRATION: High-Sensitivity Cardiac Troponin T to Optimize Chest Pain Risk Stratification (STOP-CP; ClinicalTrials.gov: NCT02984436; https://clinicaltrials.gov/ct2/show/NCT02984436).

Derivation and validation of a high sensitivity troponin-T HEART pathway.

BACKGROUND: The HEART Pathway is widely used for chest pain risk stratification but has yet to be optimized for high sensitivity troponin T (hs-cTnT) assays. METHODS: We conducted a secondary analysis of STOP-CP, a prospective cohort study enrolling adult ED patients with symptoms suggestive of acute coronary syndrome at 8 sites in the United States (US). Patients had a 0- and 1-hour hs-cTnT measured and a HEAR score completed. A derivation set consisting of 729 randomly selected participants was used to derive a hs-cTnT HEART Pathway with rule-out, observation, and rule-in groups for 30-day cardiac death or myocardial infarction (MI). Optimal baseline and 1-hour troponin cutoffs were selected using generalized cross validation to achieve a negative predictive value (NPV) >99% for rule out and positive predictive value (PPV) >60% or maximum Youden index for rule-in. Optimal 0-1-hour delta values were derived using generalized cross validation to maximize the NPV for the rule-out group and PPV for the rule-in group. The hs-cTnT HEART Pathway performance was validated in the remaining cohort (n = 723). RESULTS: Among the 1452 patients, 30-day cardiac death or MI occurred in 12.7% (184/1452). Within the derivation cohort the optimal hs-cTnT HEART Pathway classified 36.5% (266/729) into the rule-out group, yielding a NPV of 99.2% (95% CI: 98.2-100) for 30-day cardiac death or MI. The rule-in group included 15.4% (112/729) with a PPV of 55.4% (95% CI: 46.2-64.6). In the validation cohort, the hs-cTnT HEART Pathway ruled-out 37.6% (272/723), of which 2 had 30-day cardiac death or MI, yielding a NPV of 99.3% (95% CI: 98.3-100). The rule-in group included 14.5% (105/723), yielding a PPV of 57.1% (95% CI: 47.7-66.6). CONCLUSIONS: A novel hs-cTnT HEART Pathway with serial 0- and 1-hour hs-cTnT measures has high NPV and moderate PPV for 30-day cardiac death or MI.

The potential of trait data to increase the availability of bioindicators: A case study using plant conservatism values.

Biological indicators are commonly used to evaluate ecosystem condition. However, their use is often constrained by the availability of information with which to assign species-specific indicator values, which reflect species' responses to the environmental conditions being evaluated by the indicator. As these responses are driven by underlying traits, and trait data for numerous species are available in publicly accessible databases, one possible approach to approximating missing bioindicator values is through traits. We used the Floristic Quality Assessment (FQA) framework and its component indicator of disturbance sensitivity, species-specific ecological conservatism scores (C-scores), as a study system to test the potential of this approach. We tested the consistency of relationships between trait values and expert-assigned C-scores and the trait-based predictability of C-scores across five regions. Furthermore, as a proof-of-concept exercise, we used a multi-trait model to try to reconstruct C-scores, and compared the model predictions to expert-assigned scores. Out of 20 traits tested, there was evidence of regional consistency for germination rate, growth rate, propagation type, dispersal unit, and leaf nitrogen. However, the individual traits showed low predictability (R2 = 0.1-0.2) for C-scores, and a multi-trait model produced substantial classification errors; in many cases, >50% of species were misclassified. The mismatches may largely be explained by the inability to generalize regionally varying C-scores from geographically neutral/naive trait data stored in databases, and the synthetic nature of C-scores. Based on these results, we recommend possible next steps for expanding the availability of species-based bioindication frameworks such as the FQA. These steps include increasing the availability of geographic and environmental data in trait databases, incorporating data about intraspecific trait variability into these databases, conducting hypothesis-driven investigations into trait-indicator relationships, and having regional experts review our results to determine if there are patterns in the species that were correctly or incorrectly classified.

MDiGest: A Python package for describing allostery from molecular dynamics simulations.

Many biological processes are regulated by allosteric mechanisms that communicate with distant sites in the protein responsible for functionality. The binding of a small molecule at an allosteric site typically induces conformational changes that propagate through the protein along allosteric pathways regulating enzymatic activity. Elucidating those communication pathways from allosteric sites to orthosteric sites is, therefore, essential to gain insights into biochemical processes. Targeting the allosteric pathways by mutagenesis can allow the engineering of proteins with desired functions. Furthermore, binding small molecule modulators along the allosteric pathways is a viable approach to target reactions using allosteric inhibitors/activators with temporal and spatial selectivity. Methods based on network theory can elucidate protein communication networks through the analysis of pairwise correlations observed in molecular dynamics (MD) simulations using molecular descriptors that serve as proxies for allosteric information. Typically, single atomic descriptors such as α-carbon displacements are used as proxies for allosteric information. Therefore, allosteric networks are based on correlations revealed by that descriptor. Here, we introduce a Python software package that provides a comprehensive toolkit for studying allostery from MD simulations of biochemical systems. MDiGest offers the ability to describe protein dynamics by combining different approaches, such as correlations of atomic displacements or dihedral angles, as well as a novel approach based on the correlation of Kabsch-Sander electrostatic couplings. MDiGest allows for comparisons of networks and community structures that capture physical information relevant to allostery. Multiple complementary tools for studying essential dynamics include principal component analysis, root mean square fluctuation, as well as secondary structure-based analyses.

Outcomes of an integrated practice unit for vulnerable emergency department patients.

BACKGROUND: An integrated practice unit (IPU) that provides a multidisciplinary approach to patient care, typically involving a primary care provider, registered nurse, social worker, and pharmacist has been shown to reduce healthcare utilization among high-cost super-utilizer (SU) patients or multi-visit patients (MVP). However, less is known about differences in the impact of these interventions on insured vs. uninsured SU patients and super high frequency SUs ([Formula: see text]8 ED visits per 6 months) vs. high frequency SUs (4-7 ED visits per 6 months). METHODS: We assessed the percent reduction in ED visits, ED cost, hospitalizations, hospital days, and hospitalization costs following implementation of an IPU for SUs located in an academic tertiary care facility. We compared outcomes for publicly insured with uninsured patients, and super high frequency SUs with high frequency SUs 6 months before vs. 6 months after enrollment in the IPU. RESULTS: There was an overall 25% reduction in hospitalizations (p < 0.001), and 23% reduction in hospital days (p = 0.0045), when comparing 6 months before vs. 6 months after enrollment in the program. There was a 26% reduction in average total direct hospitalization costs per patient (p = 0.002). Further analysis revealed a greater reduction in health care utilization for uninsured SU patients compared with publicly insured patients. The program reduced hospitalizations for super high frequency SUs. However, there was no statistically significant impact on overall health care utilization of super high frequency SUs when compared with high frequency SUs. CONCLUSIONS: Our study supports existing evidence that dedicated IPUs for SUs can achieve significant reductions in acute care utilization, particularly for uninsured and high frequency SU patients. TRIAL REGISTRATION: IRB201500212. Retrospectively registered.

Insights into Binding of Single-Stranded Viral RNA Template to the Replication-Transcription Complex of SARS-CoV-2 for the Priming Reaction from Molecular Dynamics Simulations.

A minimal replication-transcription complex (RTC) of SARS-CoV-2 for synthesis of viral RNAs includes the nsp12 RNA-dependent RNA polymerase and two nsp8 RNA primase subunits for de novo primer synthesis, one nsp8 in complex with its accessory nsp7 subunit and the other without it. The RTC is responsible for faithfully copying the entire (+) sense viral genome from its first 5'-end to the last 3'-end nucleotides through a replication-intermediate (RI) template. The single-stranded (ss) RNA template for the RI is its 33-nucleotide 3'-poly(A) tail adjacent to a well-characterized secondary structure. The ssRNA template for viral transcription is a 5'-UUUAU-3' next to stem-loop (SL) 1'. We analyze the electrostatic potential distribution of the nsp8 subunit within the RTC around the template strand of the primer/template (P/T) RNA duplex in recently published cryo-EM structures to address the priming reaction using the viral poly(A) template. We carried out molecular dynamics (MD) simulations with a P/T RNA duplex, the viral poly(A) template, or a generic ssRNA template. We find evidence that the viral poly(A) template binds similarly to the template strand of the P/T RNA duplex within the RTC, mainly through electrostatic interactions, providing new insights into the priming reaction by the nsp8 subunit within the RTC, which differs significantly from the existing proposal of the nsp7/nsp8 oligomer formed outside the RTC. High-order oligomerization of nsp8 and nsp7 for SARS-CoV observed outside the RTC of SARS-CoV-2 is not found in the RTC and not likely to be relevant to the priming reaction.

Structural Basis for Reduced Dynamics of Three Engineered HNH Endonuclease Lys-to-Ala Mutants for the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Associated 9 (CRISPR/Cas9) Enzyme.

Many bacteria possess type-II immunity against invading phages or plasmids known as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system to detect and degrade the foreign DNA sequences. The Cas9 protein has two endonucleases responsible for double-strand breaks (the HNH domain for cleaving the target strand of DNA duplexes and RuvC domain for the nontarget strand, respectively) and a single-guide RNA-binding domain where the RNA and target DNA strands are base-paired. Three engineered single Lys-to-Ala HNH mutants (K810A, K848A, and K855A) exhibit an enhanced substrate specificity for cleavage of the target DNA strand. We report in this study that in the wild-type (wt) enzyme, D835, Y836, and D837 within the Y836-containing loop (comprising E827-D837) adjacent to the catalytic site have uncharacterizable broadened 1H15N nuclear magnetic resonance (NMR) features, whereas remaining residues in the loop have different extents of broadened NMR spectra. We find that this loop in the wt enzyme exhibits three distinct conformations over the duration of the molecular dynamics simulations, whereas the three Lys-to-Ala mutants retain only one conformation. The versatility of multiple alternate conformations of this loop in the wt enzyme could help to recruit noncognate DNA substrates into the HNH active site for cleavage, thereby reducing its substrate specificity relative to the three mutants. Our study provides further experimental and computational evidence that Lys-to-Ala substitutions reduce dynamics of proteins and thus increase their stability.

A structurally preserved allosteric site in the MIF superfamily affects enzymatic activity and CD74 activation in D-dopachrome tautomerase.

The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily.

Diagnostic Performance of High-Sensitivity Cardiac Troponin T Strategies and Clinical Variables in a Multisite US Cohort.

BACKGROUND: European data support the use of low high-sensitivity troponin (hs-cTn) measurements or a 0/1-hour (0/1-h) algorithm for myocardial infarction to exclude major adverse cardiac events (MACEs) among patients in the emergency department with possible acute coronary syndrome. However, modest US data exist to validate these strategies. This study evaluated the diagnostic performance of an initial hs-cTnT measure below the limit of quantification (LOQ: 6 ng/L), a 0/1-h algorithm, and their combination with history, ECG, age, risk factors, and initial troponin (HEART) scores for excluding MACE in a multisite US cohort. METHODS: A prospective cohort study was conducted at 8 US sites, enrolling adult patients in the emergency department with symptoms suggestive of acute coronary syndrome and without ST-elevation on ECG. Baseline and 1-hour blood samples were collected, and hs-cTnT (Roche; Basel, Switzerland) was measured. Treating providers blinded to hs-cTnT results prospectively calculated HEART scores. MACE (cardiac death, myocardial infarction, and coronary revascularization) at 30 days was adjudicated. The proportion of patients with initial hs-cTnT measures below the LOQ and risk according to a 0/1-h algorithm was determined. The negative predictive value (NPV) was calculated for both strategies when used alone or with a HEART score. RESULTS: Among 1462 participants with initial hs-cTnT measures, 46.4% (678 of 1462) were women and 37.1% (542 of 1462) were Black with an age of 57.6±12.9 (mean±SD) years. MACEs at 30 days occurred in 14.4% (210 of 1462) of participants. Initial hs-cTnT measures below the LOQ occurred in 32.8% (479 of 1462), yielding an NPV of 98.3% (95% CI, 96.7-99.3) for 30-day MACEs. A low-risk HEART score with an initial hs-cTnT below the LOQ occurred in 20.1% (294 of 1462), yielding an NPV of 99.0% (95% CI, 97.0-99.8) for 30-day MACEs. A 0/1-h algorithm was complete in 1430 patients, ruling out 57.8% (826 of 1430) with an NPV of 97.2% (95% CI, 95.9-98.2) for 30-day MACEs. Adding a low HEART score to the 0/1-h algorithm ruled out 30.8% (441 of 1430) with an NPV of 98.4% (95% CI, 96.8-99.4) for 30-day MACEs. CONCLUSIONS: In a prospective multisite US cohort, an initial hs-cTnT below the LOQ combined with a low-risk HEART score has a 99% NPV for 30-day MACEs. The 0/1-h hs-cTnT algorithm did not achieve an NPV >99% for 30-day MACEs when used alone or with a HEART score. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02984436.

Monocyte chemoattractant protein-1 is not predictive of cardiac events in patients with non-low-risk chest pain.

BACKGROUND: Prior studies suggest monocyte chemoattractant protein-1 (MCP-1) may be useful for risk stratifying ED patients with chest pain. We hypothesise that MCP-1 will be predictive of 90-day major adverse cardiovascular events (MACEs) in non-low-risk patients. METHODS: A case-control study was nested within a prospective multicentre cohort (STOP-CP), which enrolled adult patients being evaluated for acute coronary syndrome at eight US EDs from 25 January 2017 to 06 September 2018. Patients with a History, ECG, Age, and Risk factor score (HEAR score) ≥4 or coronary artery disease (CAD), a non-ischaemic ECG, and non-elevated contemporary troponins at 0 and 3 hours were included. Cases were patients with 90-day MACE (all-cause death, myocardial infarction or revascularisation). Controls were patients without MACE selected with frequency matching using age, sex, race, and HEAR score or the presence of CAD. Serum MCP-1 was measured. Sensitivity and specificity were determined for cut-off points of 194 pg/mL, 200 pg/mL, 238 pg/mL and 281 pg/mL. Logistic regression adjusting for age, sex, race, and HEAR score/presence of CAD was used to determine the association between MCP-1 and 90-day MACE. A separate logistic model also included high-sensitivity troponin (hs-cTnT). RESULTS: Among 40 cases and 179 controls, there was no difference in age (p=0.90), sex (p=1.00), race (p=0.85), or HEAR score/presence of CAD (p=0.89). MCP-1 was similar in cases (median 191.9 pg/mL, IQR: 161.8-260.1) and controls (median 196.6 pg/mL, IQR: 163.0-261.1) (p=0.48). At a cut-off point of 194 pg/mL, MCP-1 was 50.0% (95% CI 33.8% to 66.2%) sensitive and 46.9% (95% CI 39.4% to 54.5%) specific for 90-day MACE. After adjusting for covariates, MCP-1 was not associated with 90-day MACE at any cut-off point (at 194 pg/mL, OR 0.88 (95% CI 0.43 to 1.79)). When including hs-cTnT in the model, MCP-1 was not associated with 90-day MACE at any cut-off point (at 194 pg/mL, OR 0.85 (95% CI 0.42 to 1.73)). CONCLUSION: MCP-1 is not predictive of 90-day MACE in patients with non-low-risk chest pain.

Predictors of professional burnout and fulfilment in a longitudinal analysis on nurses and healthcare workers in the COVID-19 pandemic.

AIMS AND OBJECTIVES: (1) To investigate the vulnerability of nurses to experiencing professional burnout and low fulfilment across 5 months of the COVID-19 pandemic. (2) To identify modifiable variables in hospital leadership and individual vulnerabilities that may mitigate these effects. BACKGROUND: Nurses were at increased risk for burnout and low fulfilment prior to the COVID-19 pandemic. Hospital leadership factors such as organisational structure and open communication and consideration of employee opinions are known to have positive impacts on work attitudes. Personal risk factors for burnout include symptoms of depression and anxiety. METHODS: Healthcare workers (n = 406 at baseline, n = 234 longitudinal), including doctors (n = 102), nurses (n = 94), technicians (n = 90) and non-clinical administrative staff (n = 120), completed 5 online questionnaires, once per month, for 5 months. Participants completed self-report questionnaires on professional fulfilment and burnout, perceptions of healthcare leadership, and symptoms of anxiety and depression. Participants were recruited from various healthcare settings in the southeastern United States. The STROBE checklist was used to report the present study. RESULTS: Both at baseline and across the 5 months, nurses working during the COVID-19 pandemic reported increased burnout and decreased fulfilment relative to doctors. For all participants, burnout remained largely steady and fulfilment decreased slightly. The strongest predictors of both burnout and fulfilment were organisational structure and depressive symptoms. Leadership consideration and anxiety symptoms had smaller, yet significant, relationships to burnout and fulfilment in longitudinal analyses. CONCLUSIONS: Burnout and reduced fulfilment remain a problem for healthcare workers, especially nurses. Leadership styles and employee symptoms of depression and anxiety are appropriate targets for intervention. RELEVANCE TO CLINICAL PRACTICE: Leadership wishing to reduce burnout and increase fulfilment among employees should increase levels of organisational support and consideration and expand supports to employees seeking treatment for depression and anxiety.

Allosteric Impact of the Variable Insert Loop in Vaccinia H1-Related (VHR) Phosphatase.

Dynamics and conformational motions are important to the activity of enzymes, including protein tyrosine phosphatases. These motions often extend to regions outside the active site, called allosteric regions. In the tyrosine phosphatase Vaccinia H1-related (VHR) enzyme, we demonstrate the importance of the allosteric interaction between the variable insert region and the active-site loops in VHR. These studies include solution nuclear magnetic resonance, computation, steady-state, and rapid kinetic measurements. Overall, the data indicate concerted millisecond motions exist between the variable insert and the catalytic acid loop in wild-type (WT) VHR. The 150 ns computation studies show a flexible acid loop in WT VHR that opens during the simulation from its initial closed structure. Mutation of the variable insert residue, asparagine 74, to alanine results in a rigidification of the acid loop as observed by molecular dynamics simulations and a disruption of crucial active-site hydrogen bonds. Moreover, enzyme kinetic analysis shows a weakening of substrate affinity in the N74A mutant and a >2-fold decrease in substrate cleavage and hydrolysis rates. These data show that despite being nearly 20 Å from the active site, the variable insert region is linked to the acid loop by coupled millisecond motions, and that disruption of the communication between the variable insert and active site alters the normal catalytic function of VHR and perturbs the active-site environment.

Contemporary ancestor? Adaptive divergence from standing genetic variation in Pacific marine threespine stickleback.

BACKGROUND: Populations that have repeatedly colonized novel environments are useful for studying the role of ecology in adaptive divergence - particularly if some individuals persist in the ancestral habitat. Such "contemporary ancestors" can be used to demonstrate the effects of selection by comparing phenotypic and genetic divergence between the derived population and their extant ancestors. However, evolution and demography in these "contemporary ancestors" can complicate inferences about the source (standing genetic variation, de novo mutation) and pace of adaptive divergence. Marine threespine stickleback (Gasterosteus aculeatus) have colonized freshwater environments along the Pacific coast of North America, but have also persisted in the marine environment. To what extent are marine stickleback good proxies of the ancestral condition? RESULTS: We sequenced > 5800 variant loci in over 250 marine stickleback from eight locations extending from Alaska to California, and phenotyped them for platedness and body shape. Pairwise FST varied from 0.02 to 0.18. Stickleback were divided into five genetic clusters, with a single cluster comprising stickleback from Washington to Alaska. Plate number, Eda, body shape, and candidate loci showed evidence of being under selection in the marine environment. Comparisons to a freshwater population demonstrated that candidate loci for freshwater adaptation varied depending on the choice of marine populations. CONCLUSIONS: Marine stickleback are structured into phenotypically and genetically distinct populations that have been evolving as freshwater stickleback evolved. This variation complicates their usefulness as proxies of the ancestors of freshwater populations. Lessons from stickleback may be applied to other "contemporary ancestor"-derived population studies.

Genome-Wide Analysis of Alternative Splicing during Development and Drought Stress in Maize.

Alternative splicing plays a crucial role in plant development as well as stress responses. Although alternative splicing has been studied during development and in response to stress, the interplay between these two factors remains an open question. To assess the effects of drought stress on developmentally regulated splicing in maize (Zea mays), 94 RNA-seq libraries from ear, tassel, and leaf of the B73 public inbred line were constructed at four developmental stages under both well-watered and drought conditions. This analysis was supplemented with a publicly available series of 53 libraries from developing seed, embryo, and endosperm. More than 48,000 novel isoforms, often with stage- or condition-specific expression, were uncovered, suggesting that developmentally regulated alternative splicing occurs in thousands of genes. Drought induced large developmental splicing changes in leaf and ear but relatively few in tassel. Most developmental stage-specific splicing changes affected by drought were tissue dependent, whereas stage-independent changes frequently overlapped between leaf and ear. A linear relationship was found between gene expression changes in splicing factors and alternative spicing of other genes during development. Collectively, these results demonstrate that alternative splicing is strongly associated with tissue type, developmental stage, and stress condition.

A 51-year-old woman crushed by an elephant trunk.

Wild and exotic animal attacks are not common in the United States. Animal-related injuries in the United States are usually caused by dog bites, followed by cattle and horse injuries. Exotic animal attacks can occur when the animals are provoked, depressed, or housed improperly by owners. We report the case of a 51-year-old woman who sustained multiple systemic traumatic injuries after she was pinned to a fence by an elephant's trunk. Upon arrival in the emergency department, she was hypothermic with a temperature of 35.1ºC (95.1ºF), hypotensive to 94/60 mm Hg after 5 L crystalloid, tachycardic at 108 beats/min, and intubated with oxygen saturation of 100%. Tranexamic acid was administered in addition to starting a massive transfusion protocol. Injuries included bilateral multiple rib fractures, left abdominal wall degloving injury, right pneumothorax, right hemothorax, left chest wall puncture wound, grade IV splenic laceration, 3 grade III liver lacerations, retroperitoneal hematoma, and degloving injuries to bilateral posterior thighs requiring more than 30 operations. Why should an emergency physician be aware of this? Several factors need to be considered when evaluating animal-related injuries, including type, age, and sex of the animal. Multisystem traumatic injuries should be assumed when a large animal is involved. Prehospital care and transport time are vital to a patient's survival in both urban and rural settings. During the initial resuscitation, administering antibiotics tailored to the specific animal can greatly decrease risk of infection and morbidity. Additionally, tetanus immunoglobulin, tetanus toxoid, and rabies immunoglobulin and vaccine may be needed, unless the victim has been previously vaccinated.

Kernel-elastic autoencoder for molecular design.

We introduce the kernel-elastic autoencoder (KAE), a self-supervised generative model based on the transformer architecture with enhanced performance for molecular design. KAE employs two innovative loss functions: modified maximum mean discrepancy (m-MMD) and weighted reconstruction (LWCEL). The m-MMD loss has significantly improved the generative performance of KAE when compared to using the traditional Kullback-Leibler loss of VAE, or standard maximum mean discrepancy. Including the weighted reconstruction loss LWCEL, KAE achieves valid generation and accurate reconstruction at the same time, allowing for generative behavior that is intermediate between VAE and autoencoder not available in existing generative approaches. Further advancements in KAE include its integration with conditional generation, setting a new state-of-the-art benchmark in constrained optimizations. Moreover, KAE has demonstrated its capability to generate molecules with favorable binding affinities in docking applications, as evidenced by AutoDock Vina and Glide scores, outperforming all existing candidates from the training dataset. Beyond molecular design, KAE holds promise to solve problems by generation across a broad spectrum of applications.

Rapid recognition and optimal management of hemophilia in the emergency department: A quality improvement project.

Objectives: This study aimed to assess the effectiveness of a continuous quality improvement initiative at the University of Florida Health Physicians practice in reducing the time to administer factor replacement therapy (FRT) for hemophilia patients presenting with bleeding in the emergency department (ED). Methods: The study, a quasi-experimental, interventional design, was conducted between January 2020 and January 2023. The intervention, implemented in September 2021, involved training ED physicians, creating a specialized medication order set within the electronic health record (EHR), and a rapid triage system. The effectiveness was measured by comparing the time from ED arrival to factor administration before and after the intervention and benchmarking it against the National Bleeding Disorders Foundation's Medical and Scientific Advisory Council (MASAC)-recommended 1-hour timeline for factor administration. An interrupted time series (ITS) analysis with a generalized least squares model assessed the intervention's impact. Results: A total of 43 ED visits (22 pre-intervention and 21 post-intervention) were recorded. Post-intervention, the average time from ED arrival to factor administration decreased from 5.63 to 3.15 hours. There was no significant increase (27% vs. 29%) in the patients receiving factor within 1-hour of ED arrival. The ITS analysis predicted a 20-hour reduction in the average quarterly time to administer factor by the end of the study, an 84% decrease. Conclusions: The quality improvement program decreased the time to administer FRT for patients with hemophilia in the ED. However, the majority of patients did not achieve the 1-hour MASAC-recommended timeline for factor administration after ED arrival.

Sex and race differences in the performance of the European Society of Cardiology 0/1-h algorithm with high-sensitivity troponin T.

The diagnostic performance of the high-sensitivity troponin T (hs-cTnT) European Society of Cardiology (ESC) 0/1-h algorithm in sex and race subgroups of US Emergency Department (ED) patients is unclear. A pre-planned subgroup analysis of the STOP-CP cohort study was conducted. Participants with 0- and 1-h hs-cTnT measures from eight US EDs (1/2017 to 9/2018) were stratified into rule-out, observation, and rule-in zones using the hs-cTnT ESC 0/1 algorithm. The primary outcome was adjudicated 30-day cardiac death or MI. The proportion with the primary outcome in each zone was compared between subgroups with Fisher's exact tests. The negative predictive value (NPV) of the ESC 0/1 rule-out zone for 30-day CDMI was calculated and compared between subgroups using Fisher's exact tests. Of the 1422 patients enrolled, 54.2% (770/1422) were male and 58.1% (826/1422) white with a mean age of 57.6 ± 12.8 years. At 30 days, cardiac death or myocardial infarction (MI) occurred in 12.9% (183/1422) of participants. Among patients stratified to the rule-out zone, 30-day cardiac death or MI occurred in 1.1% (5/436) of women versus 2.1% (8/436) of men (p = .40) and 1.2% (4/331) of non-white patients versus 1.8% (9/490) of white patients (p = .58). The NPV for 30-day cardiac death or MI was similar among women versus men (98.9% [95% confidence interval, CI: 97.3-99.6] vs. 97.9% [95% CI: 95.9-99.1]; p = .40) and among white versus non-white patients (98.8% [95% CI: 96.9-99.7] vs. 98.2% [95% CI: 96.5-99.2]; p = .39). NPVs <99% in each subgroup suggest the hs-cTnT ESC 0/1-h algorithm may not be safe for use in US EDs. Trial Registration: High-Sensitivity Cardiac Troponin T to Optimize Chest Pain Risk Stratification (STOP-CP; ClinicalTrials.gov: NCT02984436; https://clinicaltrials.gov/ct2/show/NCT02984436).