Blood DNA methylation in post-acute sequelae of COVID-19 (PASC): a prospective cohort study.

BACKGROUND: DNA methylation integrates environmental signals with transcriptional programs. COVID-19 infection induces changes in the host methylome. While post-acute sequelae of COVID-19 (PASC) is a long-term complication of acute illness, its association with DNA methylation is unknown. No universal blood marker of PASC, superseding single organ dysfunctions, has yet been identified. METHODS: In this single centre prospective cohort study, PASC, post-COVID without PASC, and healthy participants were enrolled to investigate their symptoms association with peripheral blood DNA methylation data generated with state-of-the-art whole genome sequencing. PASC-induced quality-of-life deterioration was scored with a validated instrument, SF-36. Analyses were conducted to identify potential functional roles of differentially methylated loci, and machine learning algorithms were used to resolve PASC severity. FINDINGS: 103 patients with PASC (22.3% male, 77.7% female), 15 patients with previous COVID-19 infection but no PASC (40.0% male, 60.0% female), and 27 healthy volunteers (48.1% male, 51.9% female) were enrolled. Whole genome methylation sequencing revealed 39 differentially methylated regions (DMRs) specific to PASC, each harbouring an average of 15 consecutive positions, that differentiate patients with PASC from the two control groups. Motif analyses of PASC-regulated DMRs identify binding domains for transcription factors regulating circadian rhythm and others. Some DMRs annotated to protein coding genes were associated with changes of RNA expression. Machine learning support vector algorithm and random forest hierarchical clustering reveal 28 unique differentially methylated positions (DMPs) in the genome discriminating patients with better and worse quality of life. INTERPRETATION: Blood DNA methylation levels identify PASC, stratify PASC severity, and suggest that DNA motifs are targeted by circadian rhythm-regulating pathways in PASC. FUNDING: This project has been funded by the following agencies: NIH-AI173035 (A. Jaitovich and R. Alisch); and NIH-AG066179 (R. Alisch).

Effect of pegbelfermin on NASH and fibrosis-related biomarkers and correlation with histological response in the FALCON 1 trial.

Background & Aims: FALCON 1 was a phase IIb study of pegbelfermin in patients with non-alcoholic steatohepatitis (NASH) and stage 3 fibrosis. This FALCON 1 post hoc analysis aimed to further assess the effect of pegbelfermin on NASH-related biomarkers, correlations between histological assessments and non-invasive biomarkers, and concordance between the week 24 histologically assessed primary endpoint response and biomarkers. Methods: Blood-based composite fibrosis scores, blood-based biomarkers, and imaging biomarkers were evaluated for patients with available data from FALCON 1 at baseline through week 24. SomaSignal tests assessed protein signatures of NASH steatosis, inflammation, ballooning, and fibrosis in blood. Linear mixed-effect models were fit for each biomarker. Correlations and concordance were assessed between blood-based biomarkers, imaging, and histological metrics. Results: At week 24, pegbelfermin significantly improved blood-based composite fibrosis scores (ELF, FIB-4, APRI), fibrogenesis biomarkers (PRO-C3 and PC3X), adiponectin, CK-18, hepatic fat fraction measured by MRI-proton density fat fraction, and all four SomaSignal NASH component tests. Correlation analyses between histological and non-invasive measures identified four main categories: steatosis/metabolism, tissue injury, fibrosis, and biopsy-based metrics. Concordant and discordant effects of pegbelfermin on the primary endpoint vs. biomarker responses were observed; the most clear and concordant effects were on measures of liver steatosis and metabolism. A significant association between hepatic fat measured histologically and by imaging was observed in pegbelfermin arms. Conclusions: Pegbelfermin improved NASH-related biomarkers most consistently through improvement of liver steatosis, though biomarkers of tissue injury/inflammation and fibrosis were also improved. Concordance analysis shows that non-invasive assessments of NASH support and exceed the improvements detected by liver biopsy, suggesting that greater consideration should be given to the totality of available data when evaluating the efficacy of NASH therapeutics. Clinical trial number: Post hoc analysis of NCT03486899. Impact and implications: FALCON 1 was a study of pegbelfermin vs. placebo in patients with non-alcoholic steatohepatitis (NASH) without cirrhosis; in this study, patients who responded to pegbelfermin treatment were identified through examination of liver fibrosis in tissue samples collected through biopsy. In the current analysis, non-invasive blood- and imaging-based measures of fibrosis, liver fat, and liver injury were used to determine pegbelfermin treatment response to see how they compared with the biopsy-based results. We found that many of the non-invasive tests, particularly those that measured liver fat, identified patients who responded to pegbelfermin treatment, consistent with the liver biopsy findings. These results suggest that there may be additional value in using data from non-invasive tests, along with liver biopsy, to evaluate how well patients with NASH respond to treatment.

Sox17 mediates adult arterial endothelial cell adaptation to hemodynamics.

Sox17 is a critical regulator of arterial identity during early embryonic vascular development. However, its role in adult endothelial cells (ECs) are not fully understood. Sox17 is highly expressed in arterial ECs but not in venous ECs throughout embryonic development to adulthood suggesting that it may play a functional role in adult arteries. Here, we investigated Sox17 mediated phenotypical changes in adult ECs. To precisely control the temporal expression level of Sox17, we designed a tetracycline-inducible lentiviral gene expression system to express Sox17 selectively in cultured venous ECs. We confirmed that Sox17-induced ECs exhibit a gene profile favoring arterial and tip cell identity. Furthermore, in comparison to control ECs, Sox17-activated ECs under shear leads to greater expression of arterial markers and suppression of venous identity. These data suggest that Sox17 enables greater hemodynamic adaptability of ECs in response to fluid shear stress. Here, we also demonstrate key morphogenic behaviors of Sox17-mediated ECs. In both vasculogenic and angiogenic 3D fibrin gel studies, Sox17-mediated ECs prefer to form cohesive vessels with one another while interfering the vessel formation of the control ECs. Sox17-mediated ECs elicit hyper-sprouting behavior in the presence of pericytes but not fibroblasts, suggesting Sox17 mediated sprouting frequency is dependent on supporting cell type. Using a microfluidic chip, we also show that Sox17-mediated ECs maintain thinner diameter vessels that do not widen under interstitial flow like the control ECs. Taken together, these data showed that Sox17 mediated EC gene expression and phenotypical changes are highly modulated in the context of biomechanical stimuli, suggesting Sox17 plays a role in regulating the arterial ECs adaptability under arterial hemodynamics as well as tip cells behavior during angiogenesis and vasculogenesis. The results from this study may be valuable in improving vein graft adaptation to arterial hemodynamics and bioengineering microvasculature for tissue engineering applications.

Simulated driving performance among daily and occasional cannabis users.

OBJECTIVE: Daily cannabis users develop tolerance to some drug effects, but the extent to which this diminishes driving impairment is uncertain. This study compared the impact of acute cannabis use on driving performance in occasional and daily cannabis users using a driving simulator. METHODS: We used a within-subjects design to observe driving performance in adults age 25 to 45 years with different cannabis use histories. Eighty-five participants (43 males, 42 females) were included in the final analysis: 24 occasional users (1 to 2 times per week), 31 daily users and 30 non-users. A car-based driving simulator (MiniSim™, National Advanced Driving Simulator) was used to obtain two measures of driving performance, standard deviation of lateral placement (SDLP) and speed relative to posted speed limit, in simulated urban driving scenarios at baseline and 30 min after a 15 min ad libitum cannabis smoking period. Participants smoked self-supplied cannabis flower product (15% to 30% tetrahydrocannabinol (THC). Blood samples were collected before and after smoking (30 min after the start of smoking). Non-users performed the same driving scenarios before and after an equivalent rest interval. Changes in driving performance were analyzed by repeated measures general linear models. RESULTS: Mean whole blood THC cannabinoids concentrations post smoking were use THC = 6.4 ± 5.6 ng/ml, THC-COOH = 10.9 ± 8.79 ng/mL for occasional users and THC = 36.4 ± 37.4 ng/mL, THC-COOH = 98.1 ± 90.6 ng/mL for daily users. On a scale of 0 to 100, the mean post-use score of subjective high was similar in occasional users and daily users (52.4 and 47.2, respectively). In covariate-adjusted analysis, occasional users had a significant increase in SDLP in the straight road segment from pre to post compared to non-users; non-users decreased by a mean of 1.1 cm (25.5 cm to 24.4 cm) while occasional users increased by a mean of 1.9 cm (21.7 cm to 23.6 cm; p = 0.02). Daily users also increased adjusted SDLP in straight road segments from baseline to post-use (23.2 cm to 25.0 cm), but the change relative to non-users was not statistically significant (p = 0.08). The standardized mean difference in unadjusted SDLP from baseline to post-use in the straight road segments comparing occasional users to non-users was 0.64 (95% CI 0.09 - 1.19), a statistically significant moderate increase. When occasional users were contrasted with daily users, the baseline to post changes in SDLP were not statistically significant. Daily users exhibited a mean decrease in baseline to post-use adjusted speed in straight road segments of 1.16 mph; a significant change compared to slight speed increases in the non-users and occasional users (p = 0.02 and p = 0.01, respectively). CONCLUSION: We observed a decrement in driving performance assessed by SDLP after acute cannabis smoking that was statistically significant only in the occasional users in comparison to the nonusers. Direct contrasts between the occasional users and daily users in SDLP were not statistically significant. Daily users drove slower after cannabis use as compared to the occasional use group and non-users. The study results do not conclusively establish that occasional users exhibit more driving impairment than daily users when both smoke cannabis ad libitum.

Development and validation of a prediction model for postpartum hemorrhage at a single safety net tertiary care center.

BACKGROUND: Postpartum hemorrhage is a leading cause of pregnancy-related morbidity and mortality; however, there is limited ability to identify women at risk of this obstetrical complication. OBJECTIVE: This study aimed to develop and validate a prediction model for postpartum hemorrhage based on antenatal and intrapartum risk factors. STUDY DESIGN: This was a retrospective cohort study of women who delivered between April 2016 and March 2019 at a single safety net hospital. The prevalence of postpartum hemorrhage, defined as blood loss of ≥1000 mL at the time of delivery, was determined, and characteristics were compared between women with and without postpartum hemorrhage. Women were randomly assigned to a prediction or a validation cohort. The selection of predictors to be included in the model was based on known antenatal and intrapartum risk factors for postpartum hemorrhage. A multivariable logistic regression with a backward stepwise approach was used to create a prediction model. Area under the receiver operating characteristic curve and 95% bootstrap confidence intervals were calculated. Using the final model, a single threshold for classifying postpartum hemorrhage was chosen, and the resulting sensitivity, specificity, and false-negative and false-positive rates were explored. RESULTS: The prevalence rates of postpartum hemorrhage in the prediction and validation cohorts were 6.3% (377 of 6000 cases) and 6.4% (241 of 3774 cases), respectively (P=.83). The following predictors were selected for the final model: maternal body mass index (kg/m2), number of fetuses, history of postpartum hemorrhage, admission platelets of <100,000/µL, chorioamnionitis, arrest of descent, placental abruption, and active labor duration. The predictive model had an area under the receiver operating characteristic curve of 0.82 (95% confidence interval, 0.81-0.84). When applied to the validation cohort, the model had an area under the receiver operating characteristic curve of 0.81 (95% confidence interval, 0.78-0.83), a sensitivity of 86.9%, a specificity of 74.2%, a positive predictive value of 18.6%, a negative predictive value of 98.8%, a false-negative rate of 13.1%, and a false-positive rate of 25.9%. CONCLUSION: The model performed reasonably well in identifying women at risk of postpartum hemorrhage. Further studies are necessary to evaluate the model in clinical practice and its effect on decreasing the prevalence of postpartum hemorrhage and associated maternal morbidity.

Drug product dispensing and estimates of use in a general population survey as a signal detection problem.

PURPOSE: Understanding potential bias due to rarity of the outcome is important when monitoring newly approved drugs and drugs with low availability to the general public. Although there is an increasing use of online surveys to investigate health outcomes, the limits of inference due to drug availability have not been studied. The goal of this study was to quantify the relationship between dispensing of prescription drugs and estimates of use in an online general population survey. METHODS: An online repeated, cross-sectional survey from 2018 to 2020 was used to estimate the number of adults in the United States who used prescription drugs in the general population and compared to estimated number of prescriptions dispensed over an equivalent time period. Joinpoint regression was used to quantify thresholds. A sample of respondents was retested to estimate reliability statistics. RESULTS: A model with a single threshold was the best fit, with the estimated threshold of 565 000 (95% CI: 9500-11 600 000) prescriptions dispensed per year. Above the threshold, there was a significant association between dispensing and estimates (p < 0.001); below the threshold, the relationship was not significant (p = 0.912). Above the threshold, responses were more reliable than random chance, and reliability steadily increased with increased dispensing. CONCLUSIONS: These results suggest the threshold demarcates two distinct pharmacoepidemiological paradigms when investigating drug use in general population surveys. Dispensing can be used as a guide to determine the epidemiological paradigm that is best suited.

MEF2 (Myocyte Enhancer Factor 2) Is Essential for Endothelial Homeostasis and the Atheroprotective Gene Expression Program.

OBJECTIVE: Atherosclerosis predominantly forms in regions of oscillatory shear stress while regions of laminar shear stress are protected. This protection is partly through the endothelium in laminar flow regions expressing an anti-inflammatory and antithrombotic gene expression program. Several molecular pathways transmitting these distinct flow patterns to the endothelium have been defined. Our objective is to define the role of the MEF2 (myocyte enhancer factor 2) family of transcription factors in promoting an atheroprotective endothelium. Approach and Results: Here, we show through endothelial-specific deletion of the 3 MEF2 factors in the endothelium, Mef2a, -c, and -d, that MEF2 is a critical regulator of vascular homeostasis. MEF2 deficiency results in systemic inflammation, hemorrhage, thrombocytopenia, leukocytosis, and rapid lethality. Transcriptome analysis reveals that MEF2 is required for normal regulation of 3 pathways implicated in determining the flow responsiveness of the endothelium. Specifically, MEF2 is required for expression of Klf2 and Klf4, 2 partially redundant factors essential for promoting an anti-inflammatory and antithrombotic endothelium. This critical requirement results in phenotypic similarities between endothelial-specific deletions of Mef2a/c/d and Klf2/4. In addition, MEF2 regulates the expression of Notch family genes, Notch1, Dll1, and Jag1, which also promote an atheroprotective endothelium. In contrast to these atheroprotective pathways, MEF2 deficiency upregulates an atherosclerosis promoting pathway through increasing the amount of TAZ (transcriptional coactivator with PDZ-binding motif). CONCLUSIONS: Our results implicate MEF2 as a critical upstream regulator of several transcription factors responsible for gene expression programs that affect development of atherosclerosis and promote an anti-inflammatory and antithrombotic endothelium. Graphic Abstract: A graphic abstract is available for this article.

Agreement between a single, self-defined burnout item and the ProQOL burnout subscale for behavioural health staff.

Burnout in the field of behavioural health care is widespread. Occupational burnout can negatively impact providers' well-being and patient care, as well as lead to substantial fiscal cost to healthcare institutions. The objective of this quality improvement project was to develop a single-item survey to rapidly assess individual burnout and workforce well-being among behavioural health staff in our urban, safety-net hospital. We examined the degree of agreement between a single, self-defined burnout item from the Mini-Z and the ProQOL burnout subscale among one hundred and thirty-five nurses, behavioural technicians and administrative staff. Our findings indicate that ProQOL and Mini-Z have a low-to-moderate correlation at a baseline (k = 0.52, 95% CI 0.26, 0.69). However, using a modified ProQOL cut-off score with a binary classification of both surveys yields a moderate-to-high agreement (K = 0.67, 95% CI 0.54, 0.80). To our knowledge, this is the first published comparison of the Mini-Z with the ProQOL instrument. The project adheres to the Standards for Quality Improvement Reporting Excellence (SQUIRE) 2.0 reporting guidelines for quality improvement (Ogrinc et al., 2016). A single, validated question measuring burnout allows for more rapid assessment and the maximization of response rates, both of which are important steps in evaluating the level of burnout of the collective whole.

Tramadol non-medical use in Four European countries: A comparative analysis.

BACKGROUND: Understanding prescription medication misuse is challenging due to lack of consistent measures of misuse behaviors and prevalence between countries. Tramadol is an atypical opioid with a dual mechanism, and has low drug liking compared to conventional opioids. We evaluate tramadol misuse compared to conventional opioids utilizing a harmonized validated national survey across four countries: Germany, Italy, Spain, and the United Kingdom (UK). METHODS: Data from the Survey of Non-Medical Use of Prescription Drugs (NMURx) Program online cross-sectional general population national surveys are analyzed from 2018 from four countries, with 45,000 total responses. Misuse and abuse of tramadol, codeine, morphine, and oxycodone are compared, and national prevalence estimates calculated via calibration weighting. Rates are calculated per population and per drug availability. Supplemental data are included from patients entering treatment centres and poison centre exposures. RESULTS: In 2018, distribution, misuse, and abuse of four prescription opioids show similar patterns across four countries. In all countries, codeine is misused by the largest number of adults (estimated 861,181 in Italy to 4,676,680 in Spain in past 12 months). When adjusted for availability, tramadol is misused uncommonly with lowest or second lowest rates in all countries. Most abuse occurs by the oral route for all opioids, including tramadol with only 7.27 (Germany) to 54.92 (UK) cases per 100,000 units sold. CONCLUSIONS: In four countries, tramadol misuse and abuse are infrequent both in absolute number of cases and in comparison to conventional opioids. Even with availability of intravenous tramadol formulations, misuse by injection is rare.

The AdS5 × S5 superstring.

The duality between the type IIB superstring theory in an AdS5 × S5 background with N units of five-form flux and N = 4 super Yang-Mills theory with a U(N) gauge group has been studied extensively. My version of the construction of the superstring world-sheet action is reviewed here.

Recovery from Copperhead Snake Envenomation: Role of Age, Sex, Bite Location, Severity, and Treatment.

INTRODUCTION: Few data exist to understand the recovery phase of pit viper envenomation. A recently published placebo-controlled clinical trial affords this opportunity. The purpose of this study is to examine the time course of recovery from copperhead snake (Agkistrodon contortrix) envenomation patients managed with and without the use of antivenom, stratified by age, sex, anatomic site of envenomation, initial severity of envenomation, and geographic region. METHODS: This is a post-hoc subgroup analysis of data from a multi-center double-blinded clinical trial of Fab antivenom (FabAV) vs. placebo. Outcomes were the Patient-Specific Functional Scale (PSFS) score at 3, 7, 10, and 14 days after envenomation. Least-squares mean PSFS score curves were calculated for each subgroup, and repeated measures ANOVA was used to estimate between-group comparisons. RESULTS: Seventy-two subjects were included, of whom 44 received FabAV. Males demonstrated better overall recovery than females (model predicted PSFS score 6.18 vs 4.99; difference 1.19; 95% CI 0.12 to 2.25; p = 0.029). No sex difference was found in response to FabAV. Overall recovery and effect of FabAV were similar in adult vs adolescent patients, patients with upper vs lower extremity envenomation, and patients with initially mild vs moderate envenomation signs. Analysis by geographic location was not successful due to ANOVA mode instability. CONCLUSIONS: Male victims of copperhead snake envenomation demonstrate slightly better recovery than females, but response to Fab antivenom overall is similar across all subgroups studied.

Cardiomyocyte orientation modulated by the Numb family proteins-N-cadherin axis is essential for ventricular wall morphogenesis.

The roles of cellular orientation during trabecular and ventricular wall morphogenesis are unknown, and so are the underlying mechanisms that regulate cellular orientation. Myocardial-specific Numb and Numblike double-knockout (MDKO) hearts display a variety of defects, including in cellular orientation, patterns of mitotic spindle orientation, trabeculation, and ventricular compaction. Furthermore, Numb- and Numblike-null cardiomyocytes exhibit cellular behaviors distinct from those of control cells during trabecular morphogenesis based on single-cell lineage tracing. We investigated how Numb regulates cellular orientation and behaviors and determined that N-cadherin levels and membrane localization are reduced in MDKO hearts. To determine how Numb regulates N-cadherin membrane localization, we generated an mCherry:Numb knockin line and found that Numb localized to diverse endocytic organelles but mainly to the recycling endosome. Consistent with this localization, cardiomyocytes in MDKO did not display defects in N-cadherin internalization but rather in postendocytic recycling to the plasma membrane. Furthermore, N-cadherin overexpression via a mosaic model partially rescued the defects in cellular orientation and trabeculation of MDKO hearts. Our study unravels a phenomenon that cardiomyocytes display spatiotemporal cellular orientation during ventricular wall morphogenesis, and its disruption leads to abnormal trabecular and ventricular wall morphogenesis. Furthermore, we established a mechanism by which Numb modulates cellular orientation and consequently trabecular and ventricular wall morphogenesis by regulating N-cadherin recycling to the plasma membrane.

Quantitative PCR enumeration of vcgC and 16S rRNA type A and B genes as virulence indicators for environmental and clinical strains of Vibrio vulnificus in Galveston Bay oysters.

Oysters from a reef in Galveston Bay, Texas, USA, were screened for more virulent clinical strains versus less virulent environmental strains of Vibrio vulnificus using a combination of quantitative PCR assays for the virulence correlating gene (clinical variant, vcgC) and 16S rRNA types A and B (type A = environmental, type B = clinical). The combination of vcgC and 16S rRNA type B loci to determine clinical type strains was suitable, as indicated by the strong correlation (R2 = 0.98; p < 0.001) between these gene counts over time and their relative proportion (up to 93.8% and 94.3%, respectively) to vvhA genes used to quantify all strains of V. vulnificus. A strong seasonal shift of V. vulnificus strain types was observed. Environmental strains (16S rRNA type A) predominated from April to mid-June as salinities increased from 22 to 27 PSU (practical salinity unit) and temperatures rose 20 to 28 °C, with peak gene quantities of 16 812 ± 56 CFU/g. As temperatures increased to ≥30 °C from mid-June to September and salinities rose above 27 PSU, clinical strains (16S rRNA type B; vcgC) predominated with peak quantities 31 868 ± 287 and 32 360 ± 178 CFU/g, respectively.

All Tree Amplitudes of 6D (2,0) Supergravity: Interacting Tensor Multiplets and the K3 Moduli Space.

We present a twistorlike formula for the complete tree-level S matrix of six-dimensional (6D) (2,0) supergravity coupled to 21 Abelian tensor multiplets. This is the low-energy effective theory that corresponds to type IIB superstring theory compactified on a K3 surface. The formula is expressed as an integral over the moduli space of certain rational maps of the punctured Riemann sphere. By studying soft limits of the formula, we are able to explore the local moduli space of this theory, {[SO(5,21)]/[SO(5)×SO(21)]}. Finally, by dimensional reduction, we also obtain a new formula for the tree-level S matrix of 4D N=4 Einstein-Maxwell theory.

Causal inference for evaluating prescription opioid abuse using trend-in-trend design.

PURPOSE: One response to the opioid crisis in the United States has been the development of opioid analgesics with properties intended to reduce non-oral use. Previous evaluations of abuse in the community have relied on population averaged interrupted time series Poisson models with utilization offsets. However, competing interventions and secular trends complicate interpretation of time-series analyses. An alternative research design, trend-in-trend, accounts for heterogeneity in per capita opioid dispensing and unmeasured time-varying confounding, which provides a causal evaluation, provided that underlying assumptions are met. METHODS: Trend-in-trend can be modeled using a logistic regression framework. In logistic regression, exposure was any product-specific outpatient dispensing by three-digit ZIP code and calendar quarter, for 22 opioids. The outcome was any product-specific abuse case ascertained from poison centers and drug treatment programs, covering 94% of the US population, between July 2009 and December 2016. Product-specific odds ratios compared places without dispensing with places with any dispensing; the causal contrast represents the odds of product-specific abuse in the community given exposure. RESULTS: Dispensing of new and low-volume opioids varied considerably across the country, with no region showing high of all products. Of 22 opioids analyzed, the three with approved labeling as intended to deter abuse ranked near the lowest in both absolute (population-adjusted rates: 1.7, 0.9, and 8.2 per million people per quarter, respectively) and relative measures (trend-in-trend ORs: 1.96, 1.79, 1.69, respectively). CONCLUSIONS: Postmarketing studies of prescription opioid abuse may benefit by evolving from unadjusted surveillance rates to a causal inference approach.

Cell chirality regulates intercellular junctions and endothelial permeability.

Cell chirality is a newly discovered intrinsic property of the cell, reflecting the bias of the cell to polarize in the left-right axis. Despite increasing evidence on its substantial role in the asymmetric development of embryos, little is known about implications of cell chirality in physiology and disease. We demonstrate that cell chirality accounts for the nonmonotonic, dose-response relationship between endothelial permeability and protein kinase C (PKC) activation. The permeability of the endothelial cell layer is tightly controlled in our body, and dysregulation often leads to tissue inflammation and diseases. Our results show that low-level PKC activation is sufficient to reverse cell chirality through phosphatidylinositol 3-kinase/AKT signaling and alters junctional protein organization between cells with opposite chirality, leading to an unexpected substantial change in endothelial permeability. Our findings suggest that cell chirality regulates intercellular junctions in important ways, providing new opportunities for drug delivery across tightly connected semipermeable cellular sheets.

Carboxyhemoglobin Levels Induced by Cigarette Smoking Outdoors in Smokers.

INTRODUCTION: Non-invasive screening of carboxyhemoglobin saturation (SpCO) in the emergency department to detect occult exposure is increasingly common. The SpCO threshold to consider exposure in smokers is up to 9%. The literature supporting this cutoff is inadequate, and the impact of active smoking on SpCO saturation remains unclear. The primary objective was to characterize baseline SpCO in a cohort of smokers outdoors. Secondary objectives were to explore the impact of active smoking on SpCO and to compare SpCO between smokers and non-smokers. METHODS: This was a prospective cohort pilot study in two outdoor urban public areas in the USA, in a convenience sample of adult smokers. SpCO saturations were assessed non-invasively before, during, and 2 min after cigarette smoking with pulse CO-oximetry. Analyses included descriptive statistics, correlations, and a generalized estimating equation model. RESULTS: Eighty-five smokers had mean baseline SpCO of 2.7% (SD 2.6) and peak of 3.1% (SD 2.9), while 15 controls had SpCO 1.3% (SD 1.3). This was a significant difference. Time since last cigarette was associated with baseline SpCO, and active smoking increased mean SpCO. There was correlation among individual smokers' SpCO levels before, during, and 2 min after smoking, indicating smokers tended to maintain their baseline SpCO level. CONCLUSIONS: This study is the first to measure SpCO during active smoking in an uncontrolled environment. It suggests 80% of smokers have SpCO ≤ 5%, but potentially lends support for the current 9% as a threshold, depending on clinical context.

The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

OBJECTIVE: The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. APPROACH AND RESULTS: We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. CONCLUSIONS: These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury.