Analgesia and Sedation Use During Noninvasive Ventilation for Acute Respiratory Failure.

OBJECTIVES: To describe U.S. practice regarding administration of sedation and analgesia to patients on noninvasive ventilation (NIV) for acute respiratory failure (ARF) and to determine the association of this practice with odds of intubation or death. DESIGN: A retrospective multicenter cohort study. SETTING: A total of 1017 hospitals contributed data between January 2010 and September 2020 to the Premier Healthcare Database, a nationally representative healthcare database in the United States. PATIENTS: Adult (≥ 18 yr) patients admitted to U.S. hospitals requiring NIV for ARF. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We identified 433,357 patients on NIV of whom (26.7% [95% CI] 26.3%-27.0%) received sedation or analgesia. A total of 50,589 patients (11.7%) received opioids only, 40,646 (9.4%) received benzodiazepines only, 20,146 (4.6%) received opioids and benzodiazepines, 1.573 (0.4%) received dexmedetomidine only, and 2,639 (0.6%) received dexmedetomidine in addition to opioid and/or benzodiazepine. Of 433,357 patients receiving NIV, 50,413 (11.6%; 95% CI, 11.5-11.7%) patients underwent invasive mechanical ventilation on hospital days 2-5 or died on hospital days 2-30. Intubation was used in 32,301 patients (7.4%; 95% CI, 7.3-7.6%). Further, death occurred in 24,140 (5.6%; 95% CI, 5.5-5.7%). In multivariable analysis adjusting for relevant covariates, receipt of any medication studied was associated with increased odds of intubation or death. In inverse probability weighting, receipt of any study medication was also associated with increased odds of intubation or death (average treatment effect odds ratio 1.38; 95% CI, 1.35-1.40). CONCLUSIONS: The use of sedation and analgesia during NIV is common. Medication exposure was associated with increased odds of intubation or death. Further investigation is needed to confirm this finding and determine whether any subpopulations are especially harmed by this practice.

Routine Emergency Department Screening to Decrease Subsequent Physical Abuse.

STUDY OBJECTIVE: Emergency department (ED) screening for child physical abuse has been widely implemented, with uncertain effects on child abuse identification. Our goal was to determine the effect of screening on referrals to child protective services (CPS) identifying abuse. METHODS: We performed a retrospective cohort study of children younger than 6 years old with an ED encounter at 1 of 2 large health care systems, one of which implemented routine child abuse screening. The main outcome was initial (<2 days) or subsequent (3 to 180 days) referral to CPS identifying child abuse using linked records. We compared outcomes for the 2-year period after screening was implemented to the preperiod and nonscreening EDs using generalized estimating equations to adjust for sex, age, race/ethnicity, payor and prior ED encounters and clustered by center. RESULTS: Of the 331,120 ED encounters, 41,589 (12.6%) occurred at screening EDs during the screening period. Screening was completed in 34,272 (82%) and was positive in 188 (0.45%). Overall, 7,623 encounters (2.3%) had a subsequent referral, of which 589 (0.2%) identified moderate or severe abuse. ED screening did not change initial (adjusted odds ratio [aOR]=1.01, 95% confidence interval [CI] 0.89 to 1.15) or subsequent referral to CPS when compared to the prescreening period (aOR=1.05, 95% CI 0.9 to 1.18) or to the nonscreening EDs (aOR=1.06, 95% CI 0.92 to 1.21). CONCLUSION: Routine screening did not affect initial or subsequent referrals to CPS.

Can a Transparent Machine Learning Algorithm Predict Better than Its Black Box Counterparts? A Benchmarking Study Using 110 Data Sets.

We developed a novel machine learning (ML) algorithm with the goal of producing transparent models (i.e., understandable by humans) while also flexibly accounting for nonlinearity and interactions. Our method is based on ranked sparsity, and it allows for flexibility and user control in varying the shade of the opacity of black box machine learning methods. The main tenet of ranked sparsity is that an algorithm should be more skeptical of higher-order polynomials and interactions a priori compared to main effects, and hence, the inclusion of these more complex terms should require a higher level of evidence. In this work, we put our new ranked sparsity algorithm (as implemented in the open source R package, sparseR) to the test in a predictive model "bakeoff" (i.e., a benchmarking study of ML algorithms applied "out of the box", that is, with no special tuning). Algorithms were trained on a large set of simulated and real-world data sets from the Penn Machine Learning Benchmarks database, addressing both regression and binary classification problems. We evaluated the extent to which our human-centered algorithm can attain predictive accuracy that rivals popular black box approaches such as neural networks, random forests, and support vector machines, while also producing more interpretable models. Using out-of-bag error as a meta-outcome, we describe the properties of data sets in which human-centered approaches can perform as well as or better than black box approaches. We found that interpretable approaches predicted optimally or within 5% of the optimal method in most real-world data sets. We provide a more in-depth comparison of the performances of random forests to interpretable methods for several case studies, including exemplars in which algorithms performed similarly, and several cases when interpretable methods underperformed. This work provides a strong rationale for including human-centered transparent algorithms such as ours in predictive modeling applications.

Spiers Memorial Lecture: Shielding the active site: a streptavidin superoxide-dismutase chimera as a host protein for asymmetric transfer hydrogenation.

By anchoring a metal cofactor within a host protein, so-called artificial metalloenzymes can be generated. Such hybrid catalysts combine the versatility of transition metals in catalyzing new-to-nature reactions with the power of genetic-engineering to evolve proteins. With the aim of gaining better control over second coordination-sphere interactions between a streptavidin host-protein (Sav) and a biotinylated cofactor, we engineered a hydrophobic dimerization domain, borrowed from superoxide dismutase C (SOD), on Sav's biotin-binding vestibule. The influence of the SOD dimerization domain (DD) on the performance of an asymmetric transfer hydrogenase (ATHase) resulting from anchoring a biotinylated Cp*Ir-cofactor - [Cp*Ir(biot-p-L)Cl] (1-Cl) - within Sav-SOD is reported herein. We show that, depending on the nature of the residue at position Sav S112, the introduction of the SOD DD on the biotin-binding vestibule leads to an inversion of configuration of the reduction product, as well as a fivefold increase in catalytic efficiency. The findings are rationalized by QM/MM calculations, combined with X-ray crystallography.

Pattern-based clustering of daily weigh-in trajectories using dynamic time warping.

"Smart"-scales are a new tool for frequent monitoring of weight change as well as weigh-in behavior. These scales give researchers the opportunity to discover patterns in the frequency that individuals weigh themselves over time, and how these patterns are associated with overall weight loss. Our motivating data come from an 18-month behavioral weight loss study of 55 adults classified as overweight or obese who were instructed to weigh themselves daily. Adherence to daily weigh-in routines produces a binary times series for each subject, indicating whether a participant weighed in on a given day. To characterize weigh-in by time-invariant patterns rather than overall adherence, we propose using hierarchical clustering with dynamic time warping (DTW). We perform an extensive simulation study to evaluate the performance of DTW compared to Euclidean and Jaccard distances to recover underlying patterns in adherence time series. In addition, we compare cluster performance using cluster validation indices (CVIs) under the single, average, complete, and Ward linkages and evaluate how internal and external CVIs compare for clustering binary time series. We apply conclusions from the simulation to cluster our real data and summarize observed weigh-in patterns. Our analysis finds that the adherence trajectory pattern is significantly associated with weight loss.

Standardization of Trauma, General Surgical Morbidity and Mortality Conferences: Development and Dissemination of a "Toolkit" in Peru.

BACKGROUND: Morbidity and Mortality (M&M) conferences allow clinicians to review adverse events and identify areas for improvement. There are few reports of structured M&M conferences in low- and middle-income countries and no report of collaborative efforts to standardize them. METHODS: The present study aims to gather general surgeons representing most of Peru's urban surgical care and, in collaboration, with trauma quality improvement experts develop a M&M conferences toolkit with the expectation that its diffusion impacts their reported clinical practice. Fourteen general surgeons developed a toolkit as part of a working group under the auspices of the Peruvian General Surgery Society. After three years, we conducted an anonymous written questionnaire to follow-up previous observations of quality improvement practices. RESULTS: A four-component toolkit was developed: Toolkit component #1: Conference logistics and case selection; Toolkit component #2: Documenting form; Toolkit component #3: Presentation template; and Toolkit component #4: Code of conduct. The toolkit was disseminated to 10 hospitals in 2016. Its effectiveness was evaluated by comparing the results of surveys on quality improvement practices conducted in 2016, before toolkit dissemination (101 respondents) and 2019 (105 respondents). Lower attendance was reported by surgeons in 2019. However, in 2019, participants more frequently described "improve the system" as the perceived objective of M&M conferences (70.5% vs. 38.6% in 2016; p < 0.001). CONCLUSION: We established a toolkit for the national dissemination of a standardized M&M conference. Three years following the initial assessment in Peru, we found similar practice patterns except for increased reporting of "system improvement" as the goal of M&M conferences.

Differences in L-arginine metabolism and asthma morbidity among asthma patients with and without obstructive sleep apnea.

BACKGROUND: Imbalance in L-arginine and nitric oxide (NO) metabolism has been implicated in the pathophysiology of asthma and obstructive sleep apnea (OSA), and both diseases impact the other's morbidity. We sought to determine whether L-arginine/NO metabolism differs between adults with asthma with or without comorbid OSA, and its association with asthma morbidity. METHODS: This is a cross-sectional study of 322 adults with asthma recruited in Denver, CO and New York City, NY. Data were collected on OSA status, spirometry, and metrics of asthma control and morbidity. L-Arginine metabolites were quantified in patient serum. Bivariate analyses and multiple regression were performed to determine differences between L-arginine metabolism, OSA and association with asthma morbidity. RESULTS: Among the 322 participants, 92 (28.5%) had OSA. The cohort was 81.6% female, 23.4% identified as Black and 30.6% as Latino. Patients with asthma and OSA had significantly higher serum concentrations of NO synthase inhibitor asymmetric dimethylarginine (ADMA) (p-value = 0.019), lower L-arginine to ornithine ratios (p-value = 0.003), and increased ornithine (p-value = 0.001) and proline levels (p-value < 0.001) compared to those without OSA. In adjusted models, OSA was associated with worse asthma control, adjusted mean difference in asthma control questionnaire of 0.36 (95% confidence interval [CI]: 0.06 to 0.65), and asthma quality of life questionnaire, adjusted mean difference: - 0.53 (95% CI: - 0.85 to - 0.21), after adjusting for relevant covariates including body mass index and L-arginine metabolites. CONCLUSIONS: Adults with asthma and OSA had increased ADMA, an inhibitor of nitric oxide synthase, and greater metabolism of L-arginine via the arginase pathway compared to those with asthma alone, indicating a possible shared pathophysiological mechanism of these diseases.

Voluntary, temporary out-of-home firearm storage: A survey of firearm retailers and ranges in two states.

Out-of-home storage of personal firearms is one recommended option for individuals at risk of suicide, and statewide online maps of storage locations have been created in multiple states, including Colorado and Washington. We sought to examine both the extent to which firearm retailers and ranges offer temporary, voluntary firearm storage and the perceived barriers to providing this service. We invited all firearm retailers and ranges in Colorado and Washington to complete an online or mailed survey; eligible sites had to have a physical location where they could provide storage. Between June-July 2021, 137 retailers/ranges completed the survey (response rate = 25.1%). Nearly half (44.5%) of responding firearm retailers/ranges in Colorado and Washington State indicated they had ever provided firearm storage. Among those who had ever offered storage, 80.3% currently offered storage while 19.7% no longer did. The majority (68.6%) of participants had not heard of the Colorado/Washington gun storage maps and 82.5% did not believe they were currently listed on the maps. Respondents indicated liability waivers would most influence their decision about whether to start or continue providing temporary, voluntary storage of firearms. Understanding current practices, barriers, and concerns about providing out-of-home storage by retailers and ranges may support development of more feasible approaches for out-of-home firearm storage during times of suicide risk.

Inference following multiple imputation for generalized additive models: an investigation of the median p-value rule with applications to the Pulmonary Hypertension Association Registry and Colorado COVID-19 hospitalization data.

BACKGROUND: Missing data prove troublesome in data analysis; at best they reduce a study's statistical power and at worst they induce bias in parameter estimates. Multiple imputation via chained equations is a popular technique for dealing with missing data. However, techniques for combining and pooling results from fitted generalized additive models (GAMs) after multiple imputation have not been well explored. METHODS: We simulated missing data under MCAR, MAR, and MNAR frameworks and utilized random forest and predictive mean matching imputation to investigate a variety of rules for combining GAMs after multiple imputation with binary and normally distributed outcomes. We compared multiple pooling procedures including the "D2" method, the Cauchy combination test, and the median p-value (MPV) rule. The MPV rule involves simply computing and reporting the median p-value across all imputations. Other ad hoc methods such as a mean p-value rule and a single imputation method are investigated. The viability of these methods in pooling results from B-splines is also examined for normal outcomes. An application of these various pooling techniques is then performed on two case studies, one which examines the effect of elevation on a six-minute walk distance (a normal outcome) for patients with pulmonary arterial hypertension, and the other which examines risk factors for intubation in hospitalized COVID-19 patients (a dichotomous outcome). RESULTS: In comparison to the results from generalized additive models fit on full datasets, the median p-value rule performs as well as if not better than the other methods examined. In situations where the alternative hypothesis is true, the Cauchy combination test appears overpowered and alternative methods appear underpowered, while the median p-value rule yields results similar to those from analyses of complete data. CONCLUSIONS: For pooling results after fitting GAMs to multiply imputed datasets, the median p-value is a simple yet useful approach which balances both power to detect important associations and control of Type I errors.

Imaging Intracranial Aneurysms in the Endovascular Era: Surveillance and Posttreatment Follow-up.

While most intracranial aneurysms (IAs) remain asymptomatic over a patient's lifetime, those that rupture can cause devastating outcomes. The increased usage and quality of neuroimaging has increased detection of unruptured IAs and driven an increase in surveillance and treatment of these lesions. Standard practice is to treat incidentally discovered unruptured IAs that confer high rupture risk as well as ruptured IAs to prevent rehemorrhage. IAs are increasingly treated with coil embolization instead of microsurgical clipping; more recently, flow diversion and intrasaccular flow disruption have further expanded the versatility and utility of endovascular IA treatment. Imaging is increasingly used for posttreatment IA follow-up in the endovascular era. While cerebral angiography remains the standard for IA characterization and treatment planning, advances in CT and CT angiography and MR angiography have improved the diagnostic accuracy of noninvasive imaging for initial diagnosis and surveillance. IA features including size, dome-to-neck ratio, location, and orientation allow rupture risk stratification and determination of optimal treatment strategy and timing. The radiologist should be familiar with the imaging appearance of common IA treatment devices and the expected imaging findings following treatment. In distinction to clipping and coil embolization, flow diversion and intrasaccular flow disruption induce progressive aneurysm obliteration over months to years. Careful assessment of the device; the treated IA; adjacent brain, bone, meninges; and involved extracranial and intracranial vasculature is crucial at posttreatment follow-up imaging to confirm aneurysm obliteration and identify short-term and long-term posttreatment complications. An invited commentary by Chatterjee is available online. Online supplemental material and the slide presentation from the RSNA Annual Meeting are available for this article. ©RSNA, 2022.

A paleodemographic assessment of mortality and fertility rates during the second demographic transition in rural central Indiana.

OBJECTIVES: Since its inception, skeletally based paleodemographic research has emphasized the utility of biocultural models for interpreting the dynamic relationship between the sociocultural and ecological forces accompanying demographic transitions and shaping populations' health and well-being. While the demographic transition associated with the Neolithic Revolution has been a common focus in bioarcheology, the present study analyzes human skeletal remains from a large 19th century cemetery in central Indiana to examine population dynamics during the second demographic transition, a period generally characterized by decreasing fertility rates and improvements in life expectancy. This study demonstrates the potential to methodologically identify regional variations in the timing and interactions between broad-scale socioeconomic changes and technological advancements that characterized the time period through observed changes in survivorship and fertility based on age-at-death distributions. MATERIALS AND METHODS: This study uses three temporally distinct samples (AD 1827-1869; 1870-1889; 1890-1935) from the Bethel Cemetery (n = 503). Kaplan-Meier survival analyses with a log- rank tests are utilized to evaluate survivorship and mortality over time. Next, Cox proportional hazard analyses are employed to examine the interaction between sex and time as covariates. Finally, the D0-14/D ratio is applied to estimate fertility for each of the three temporally bounded cohorts. RESULTS: The Kaplan-Meier survival analyses and Cox proportional hazard modeling revealed statistically significant differences in survivorship between the three time periods. Age-specific mortality rates are reduced among adult female and male age classes in this rural community over the course of the 19th and early 20th centuries, resulting in the increasing life expectancies associated with the second demographic transition. While mortality in early adulthood was common during the first time period and decreases thereafter, sex was not identified as a meaningful covariate. The proportion of juveniles in the three temporal samples indicate that fertility rates were higher than national averages for the better part of the 19th century and subsequently declined around the turn of 20th century for this community. CONCLUSIONS: The results indicate temporal differences between the three periods, demonstrating increased survivorship and decreased mortality and fertility over time. These findings corroborate two key features of the second demographic transition characterized by the move from high rates of both fertility and mortality to reduced rates and a general easing of demographic pressures. The observed trends likely reflect improvements in health, coinciding the industrial advance and economic development within and around Indianapolis. While the socioeconomic factors characterizing the Industrial Revolution drove demographic shifts that parallel an equally important epidemiological transition, potential regional differences are discussed to highlight variability in the timing of demographic transitions. The paleodemographic methods utilized in this study demonstrate improved accuracy and efficacy, which ultimately advances researchers' potential to disentangle population-specific socioeconomic factors that may contribute to asymmetrical experiences of health and mortality.

PRES and RCVS: Two Distinct Entities or a Spectrum of the Same Disease?

OBJECTIVES: To report a case of a patient with overlapping posterior reversible encephalopathy syndrome (PRES) and reversible cerebral vasoconstriction syndrome (RCVS), and review the existing literature emphasizing the pathophysiological overlap of these two entities. MATERIALS AND METHODS: We conducted a literature search in electronic database PubMed identifying studies reporting the overlap of PRES and RCVS. RESULTS: PRES and RCVS are two increasingly recognized entities that share similar clinical and imaging features. PRES is characterized by vasogenic edema predominantly in the parieto-occipital regions, associated with acute onset of neurological symptoms including encephalopathy, seizures, headaches, and visual disturbances. RCVS is characterized by reversible segmental and multifocal vasoconstriction of the cerebral arteries and classically presents with thunderclap headache, with or without associated focal neurological deficits and seizures. PRES is frequently associated with uncontrolled hypertension but can also be seen in the setting of renal failure, exposure to cytotoxic agents, or pre-eclampsia. RCVS is often triggered by exposure to vasoactive agents, postpartum state, or immunosuppression. We report a case of a patient presenting with vision changes and hemiparesis, and found to have extensive cytotoxic and vasogenic edema involving the cortex and subcortical white matter on brain imaging. These changes were primarily noted in the parieto-occipital and brainstem regions, along with features of reversible vasculopathy on vascular imaging suggestive of coexisting PRES and RCVS. CONCLUSIONS: PRES and RCVS share precipitating factors, clinical and radiological features, and frequently co-exist, suggesting a common pathophysiological mechanism related to reversible dysregulation of cerebral vasculature, endothelial dysfunction, and breakdown of the blood-brain barrier.

Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens.

Copper (Cu)-only superoxide dismutases (SOD) represent a newly characterized class of extracellular SODs important for virulence of several fungal pathogens. Previous studies of the Cu-only enzyme SOD5 from the opportunistic fungal pathogen Candida albicans have revealed that the active-site structure and Cu binding of SOD5 strongly deviate from those of Cu/Zn-SODs in its animal hosts, making Cu-only SODs a possible target for future antifungal drug design. C. albicans also expresses a Cu-only SOD4 that is highly similar in sequence to SOD5, but is poorly characterized. Here, we compared the biochemical, biophysical, and cell biological properties of C. albicans SOD4 and SOD5. Analyzing the recombinant proteins, we found that, similar to SOD5, Cu-only SOD4 can react with superoxide at rates approaching diffusion limits. Both SODs were monomeric and they exhibited similar binding affinities for their Cu cofactor. In C. albicans cultures, SOD4 and SOD5 were predominantly cell wall proteins. Despite these similarities, the SOD4 and SOD5 genes strongly differed in transcriptional regulation. SOD5 was predominantly induced during hyphal morphogenesis, together with a fungal burst in reactive oxygen species. Conversely, SOD4 expression was specifically up-regulated by iron (Fe) starvation and controlled by the Fe-responsive transcription factor SEF1. Interestingly, Candida tropicalis and the emerging fungal pathogen Candida auris contain a single SOD5-like SOD rather than a pair, and in both fungi, this SOD was induced by Fe starvation. This unexpected link between Fe homeostasis and extracellular Cu-SODs may help many fungi adapt to Fe-limited conditions of their hosts.

Automated Processing of Head CT Perfusion Imaging for Ischemic Stroke Triage: A Practical Guide to Quality Assurance and Interpretation.

Recent successful trials of thrombectomy launched a shift to imaging-based patient selection for stroke intervention. Many centers have adopted CT perfusion imaging (CTP) as a routine part of stroke workflow, and the demand for emergent CTP interpretation is growing. Fully automated CTP postprocessing software that rapidly generates standardized color-coded CTP summary maps with minimal user input and with easy accessibility of the software output is increasingly being adopted. Such automated postprocessing greatly streamlines clinical workflow and CTP interpretation for radiologists and other frontline physicians. However, the straightforward interface overshadows the computational complexity of the underlying postprocessing workflow, which, if not carefully examined, predisposes the interpreting physician to diagnostic errors. Using case examples, this article aims to familiarize the general radiologist with interpreting automated CTP software data output in the context of contemporary stroke management, providing a discussion of CTP acquisition and postprocessing, a stepwise guide for CTP quality assurance and troubleshooting, and a framework for avoiding clinically significant pitfalls of CTP interpretation in commonly encountered clinical scenarios. Interpreting radiologists should apply the outlined approach for quality assurance and develop a comprehensive search pattern for the identified pitfalls, to ensure accurate CTP interpretation and optimize patient selection for reperfusion.

Exploiting the vulnerable active site of a copper-only superoxide dismutase to disrupt fungal pathogenesis.

Copper-only superoxide dismutases (SODs) represent a new class of SOD enzymes that are exclusively extracellular and unique to fungi and oomycetes. These SODs are essential for virulence of fungal pathogens in pulmonary and disseminated infections, and we show here an additional role for copper-only SODs in promoting survival of fungal biofilms. The opportunistic fungal pathogen Candida albicans expresses three copper-only SODs, and deletion of one of them, SOD5, eradicated candidal biofilms on venous catheters in a rodent model. Fungal copper-only SODs harbor an irregular active site that, unlike their Cu,Zn-SOD counterparts, contains a copper co-factor unusually open to solvent and lacks zinc for stabilizing copper binding, making fungal copper-only SODs highly vulnerable to metal chelators. We found that unlike mammalian Cu,Zn-SOD1, C. albicans SOD5 indeed rapidly loses its copper to metal chelators such as EDTA, and binding constants for Cu(II) predict that copper-only SOD5 has a much lower affinity for copper than does Cu,Zn-SOD1. We screened compounds with a variety of indications and identified several metal-binding compounds, including the ionophore pyrithione zinc (PZ), that effectively inhibit C. albicans SOD5 but not mammalian Cu,Zn-SOD1. We observed that PZ both acts as an ionophore that promotes uptake of toxic metals and inhibits copper-only SODs. The pros and cons of a vulnerable active site for copper-only SODs and the possible exploitation of this vulnerability in antifungal drug design are discussed.

Enantioselective Hydroxylation of Benzylic C(sp3)-H Bonds by an Artificial Iron Hydroxylase Based on the Biotin-Streptavidin Technology.

The selective hydroxylation of C-H bonds is of great interest to the synthetic community. Both homogeneous catalysts and enzymes offer complementary means to tackle this challenge. Herein, we show that biotinylated Fe(TAML)-complexes (TAML = Tetra Amido Macrocyclic Ligand) can be used as cofactors for incorporation into streptavidin to assemble artificial hydroxylases. Chemo-genetic optimization of both cofactor and streptavidin allowed optimizing the performance of the hydroxylase. Using H2O2 as oxidant, up to ∼300 turnovers for the oxidation of benzylic C-H bonds were obtained. Upgrading the ee was achieved by kinetic resolution of the resulting benzylic alcohol to afford up to >98% ee for (R)-tetralol. X-ray analysis of artificial hydroxylases highlights critical details of the second coordination sphere around the Fe(TAML) cofactor.

Artificial Metalloenzymes Based on the Biotin-Streptavidin Technology: Enzymatic Cascades and Directed Evolution.

Artificial metalloenzymes (ArMs) result from anchoring a metal-containing moiety within a macromolecular scaffold (protein or oligonucleotide). The resulting hybrid catalyst combines attractive features of both homogeneous catalysts and enzymes. This strategy includes the possibility of optimizing the reaction by both chemical (catalyst design) and genetic means leading to achievement of a novel degree of (enantio)selectivity, broadening of the substrate scope, or increased activity, among others. In the past 20 years, the Ward group has exploited, among others, the biotin-(strept)avidin technology to localize a catalytic moiety within a well-defined protein environment. Streptavidin has proven versatile for the implementation of ArMs as it offers the following features: (i) it is an extremely robust protein scaffold, amenable to extensive genetic manipulation and mishandling, (ii) it can be expressed in E. coli to very high titers (up to >8 g·L-1 in fed-batch cultures), and (iii) the cavity surrounding the biotinylated cofactor is commensurate with the size of a typical metal-catalyzed transition state. Relying on a chemogenetic optimization strategy, varying the orientation and the nature of the biotinylated cofactor within genetically engineered streptavidin, 12 reactions have been reported by the Ward group thus far. Recent efforts within our group have focused on extending the ArM technology to create complex systems for integration into biological cascade reactions and in vivo. With the long-term goal of complementing in vivo natural enzymes with ArMs, we summarize herein three complementary research lines: (i) With the aim of mimicking complex cross-regulation mechanisms prevalent in metabolism, we have engineered enzyme cascades, including cross-regulated reactions, that rely on ArMs. These efforts highlight the remarkable (bio)compatibility and complementarity of ArMs with natural enzymes. (ii) Additionally, multiple-turnover catalysis in the cytoplasm of aerobic organisms was achieved with ArMs that are compatible with a glutathione-rich environment. This feat is demonstrated in HEK-293T cells that are engineered with a gene switch that is upregulated by an ArM equipped with a cell-penetrating module. (iii) Finally, ArMs offer the fascinating prospect of "endowing organometallic chemistry with a genetic memory." With this goal in mind, we have identified E. coli's periplasmic space and surface display to compartmentalize an ArM, while maintaining the critical phenotype-genotype linkage. This strategy offers a straightforward means to optimize by directed evolution the catalytic performance of ArMs. Five reactions have been optimized following these compartmentalization strategies: ruthenium-catalyzed olefin metathesis, ruthenium-catalyzed deallylation, iridium-catalyzed transfer hydrogenation, dirhodium-catalyzed cyclopropanation and carbene insertion in C-H bonds. Importantly, >100 turnovers were achieved with ArMs in E. coli whole cells, highlighting the multiple turnover catalytic nature of these systems.

Presence of any degree of coronary artery disease among liver transplant candidates is associated with increased rate of post-transplant major adverse cardiac events.

The impact of coronary artery disease (CAD) among liver transplant candidates (LTC) on post-LT clinical outcomes remains unclear. The aim of this study is to determine association of presence and severity of CAD on post-LT major adverse cardiac events (MACE) including cardiac-associated mortality. We conducted a retrospective cohort analysis of 231 patients who underwent diagnostic coronary angiogram (DCA) during their LT evaluation at a tertiary medical center from 2012-2017. Patients were analyzed based on degree of CAD (no CAD, non-obstructive CAD [< 50% stenosis], obstructive CAD [≥50% stenosis]) per DCA results. MACE were noted at 30 days, 1 year, 3 years, and 5 years post-LT, and Kaplan-Meier curves were used to determine post-LT MACE-free probability. LTC with any CAD, including non-obstructive CAD, had lower MACE-free probability at all post-LT time points (0.94 vs 0.65 at 30 days, P = .001; 0.87 vs 0.59 at 1 year, P = .002; 0.87 vs 0.41 at 3 years, P < .001; 0.87 vs 0.37 at 5 years, P < .001). Identification of and medical intervention for non-obstructive CAD should be considered in all LTC, though further studies are necessary to determine optimal medical interventions to mitigate MACE risk in this cohort.

Eukaryotic copper-only superoxide dismutases (SODs): A new class of SOD enzymes and SOD-like protein domains.

The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria (i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.

Skull Base-related Lesions at Routine Head CT from the Emergency Department: Pearls, Pitfalls, and Lessons Learned.

Routine non-contrast material-enhanced head CT is one of the most frequently ordered studies in the emergency department. Skull base-related pathologic entities, often depicted on the first or last images of a routine head CT study, can be easily overlooked in the emergency setting if not incorporated in the interpreting radiologist's search pattern, as the findings can be incompletely imaged. Delayed diagnosis, misdiagnosis, or lack of recognition of skull base pathologic entities can negatively impact patient care. This article reviews and illustrates the essential skull base anatomy and common blind spots that are important to radiologists who interpret nonenhanced head CT images in the acute setting. The imaging characteristics of important "do not miss" lesions are emphasized and categorized by their cause and location within the skull base, and the potential differential diagnoses are discussed. An interpretation checklist to improve diagnostic accuracy is provided. ©RSNA, 2019.