Type I interferon induces TCR-dependent and -independent antimicrobial responses in γδ intraepithelial lymphocytes.

Intraepithelial lymphocytes (IEL) expressing the γδ T cell receptor (TCR) survey the intestinal epithelium to limit the invasion of microbial pathogens. The production of type I interferon (IFN) is a central component of an antiviral immune response, yet how these pro-inflammatory cytokines contribute to γδ IEL effector function remains unclear. Based on the unique activation status of IELs, and their ability to bridge innate and adaptive immunity, we investigated the extent to which type I IFN signaling modulates γδ IEL function. Using an ex vivo culture model, we find that type I IFN alone is unable to drive IFNγ production, yet low level TCR activation synergizes with type I IFN to induce IFNγ production in murine γδ IELs. Further investigation into the underlying molecular mechanisms of co-stimulation revealed that TCRγδ-mediated activation of NFAT and JNK is required for type I IFN to promote IFNγ expression in a STAT4- dependent manner. Whereas type I IFN rapidly upregulates antiviral gene expression independent of a basal TCRγδ signal, neither tonic TCR triggering nor the presence of a TCR agonist was sufficient to elicit type I IFN-induced IFNγ production in vivo . However, bypassing proximal TCR signaling events synergized with IFNAR/STAT4 activation to induce γδ IEL IFNγ production. These findings indicate that γδ IELs contribute to host defense in response to type I IFN by mounting a rapid antimicrobial response independent of TCRγδ signaling, and under permissive conditions, produce IFNγ in a TCR-dependent manner.

Observed rates of surgical instrument errors point to visualization tasks as being a critically vulnerable point in sterile processing and a significant cause of lost chargeable OR minutes.

BACKGROUND: The reporting of surgical instrument errors historically relies on cumbersome, non-automated, human-dependent, data entry into a computer database that is not integrated into the electronic medical record. The limitations of these reporting systems make it difficult to accurately estimate the negative impact of surgical instrument errors on operating room efficiencies. We set out to determine the impact of surgical instrument errors on a two-hospital healthcare campus using independent observers trained in the identification of Surgical Instrument Errors. METHODS: This study was conducted in the 7 pediatric ORs at an academic healthcare campus. Direct observations were conducted over the summer of 2021 in the 7 pediatric ORs by 24 trained student observers during elective OR days. Surgical service line, error type, case type (inpatient or outpatient), and associated length of delay were recorded. RESULTS: There were 236 observed errors affecting 147 individual surgical cases. The three most common errors were Missing+ (n = 160), Broken/poorly functioning instruments (n = 44), and Tray+ (n = 13). Errors arising from failures in visualization (i.e. inspection, identification, function) accounted for 88.6% of all errors (Missing+/Broken/Bioburden). Significantly more inpatient cases (42.73%) had errors than outpatient cases (22.32%) (p = 0.0129). For cases in which data was collected on whether an error caused a delay (103), over 50% of both IP and OP cases experienced a delay. The average length of delays per case was 10.16 min. The annual lost charges in dollars for surgical instrument associated delays in chargeable minutes was estimated to be between $6,751,058.06 and $9,421,590.11. CONCLUSIONS: These data indicate that elimination of surgical instrument errors should be a major target of waste reduction. Most observed errors (88.6%) have to do with failures in the visualization required to identify, determine functionality, detect the presence of bioburden, and assemble instruments into the correct trays. To reduce these errors and associated waste, technological advances in instrument identification, inspection, and assembly will need to be made and applied to the process of sterile processing.

Dynamic microenvironments shape nuclear organization and gene expression.

Live imaging has revealed that the regulation of gene expression is largely driven by transient interactions. For example, many regulatory proteins bind chromatin for just seconds, and loop-like genomic contacts are rare and last only minutes. These discoveries have been difficult to reconcile with our canonical models that are predicated on stable and hierarchical interactions. Proteomic microenvironments that concentrate nuclear factors may explain how brief interactions can still mediate gene regulation by creating conditions where reactions occur more frequently. Here, we summarize new imaging technologies and recent discoveries implicating microenvironments as a potential driver of nuclear function. Finally, we propose that key properties of proteomic microenvironments, such as their size, enrichment, and lifetimes, are directly linked to regulatory function.

Decreased Left Atrial Cardiomyocyte FGF13 Expression Increases Vulnerability to Postoperative Atrial Fibrillation in Humans.

Postoperative atrial fibrillation (POAF) is the most common complication after cardiac surgery and a significant cause of increased morbidity and mortality. The development of novel POAF therapeutics has been limited by an insufficient understanding of molecular mechanisms promoting atrial fibrillation. In this observational cohort study, we enrolled 28 patients without a history of atrial fibrillation that underwent mitral valve surgery for degenerative mitral regurgitation and obtained left atrial tissue samples along the standard atriotomy incision in proximity to the right pulmonary veins. We isolated cardiomyocytes and performed transcriptome analyses demonstrating 13 differentially expressed genes associated with new-onset POAF. Notably, decreased expression of fibroblast growth factor 13 (FGF13), a fibroblast growth factor homologous factor known to modulate voltage-gated sodium channel Na V 1.5 inactivation, had the most significant association with POAF. To assess the functional significance of decreased FGF13 expression in atrial myocytes, we performed patch clamp experiments on neonatal rat atrial myocytes after siRNA-mediated FGF13 knockdown, demonstrating action potential prolongation. These critical findings indicate that decreased FGF13 expression promotes vulnerability to POAF.

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms.

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.

The ftz upstream element drives late ftz stripes but is not required for regulation of Ftz target genes.

The regulation of gene expression in precise, rapidly changing spatial patterns is essential for embryonic development. Multiple enhancers have been identified for the evolving expression patterns of the cascade of Drosophila segmentation genes that establish the basic body plan of the fly. Classic reporter transgene experiments identified multiple cis-regulatory elements (CREs) that are sufficient to direct various aspects of the evolving expression pattern of the pair-rule gene fushi tarazu (ftz). These include enhancers that coordinately activate expression in all seven stripes and stripe-specific elements that activate expression in one or more ftz stripes. Of the two 7-stripe enhancers, analysis of reporter transgenes demonstrated that the upstream element (UPS) is autoregulatory, requiring direct binding of Ftz protein to direct striped expression. Here, we asked about the endogenous role of the UPS by precisely deleting this 7-stripe enhancer. In ftzΔUPS7S homozygotes, ftz stripes appear in the same order as wildtype, and all but stripe 4 are expressed at wildtype levels by the end of the cellular blastoderm stage. This suggests that the zebra element and UPS harbor information to direct stripe 4 expression, although previous deletion analyses failed to identify a stripe-specific CRE within these two 7-stripe enhancers. However, the UPS is necessary for late ftz stripe expression, with all 7 stripes decaying earlier than wildtype in ftzΔUPS7S homozygotes. Despite this premature loss of ftz expression, downstream target gene regulation proceeds as in wildtype, and segmentation is unperturbed in the overwhelming majority of animals. We propose that this late-acting enhancer provides a buffer against perturbations in gene expression but is not required for establishment of Ftz cell fates. Overall, our results demonstrate that multiple enhancers, each directing distinct aspects of an overall gene expression pattern, contribute to fine-tuning the complex patterns necessary for embryonic development.

An integrated organoid omics map extends modeling potential of kidney disease.

Kidney organoids are a promising model to study kidney disease, but their use is constrained by limited knowledge of their functional protein expression profile. Here, we define the organoid proteome and transcriptome trajectories over culture duration and upon exposure to TNFα, a cytokine stressor. Older organoids increase deposition of extracellular matrix but decrease expression of glomerular proteins. Single cell transcriptome integration reveals that most proteome changes localize to podocytes, tubular and stromal cells. TNFα treatment of organoids results in 322 differentially expressed proteins, including cytokines and complement components. Transcript expression of these 322 proteins is significantly higher in individuals with poorer clinical outcomes in proteinuric kidney disease. Key TNFα-associated protein (C3 and VCAM1) expression is increased in both human tubular and organoid kidney cell populations, highlighting the potential for organoids to advance biomarker development. By integrating kidney organoid omic layers, incorporating a disease-relevant cytokine stressor and comparing with human data, we provide crucial evidence for the functional relevance of the kidney organoid model to human kidney disease.

Binding and transport of LPS occurs through the coordinated combination of an array of sites across the entire Escherichia coli LPS transport protein LptA.

The outer leaflet of the outer membrane (OM) of bacteria such as Escherichia coli, Pseudomonas aeruginosa, and other important pathogens is largely composed of lipopolysaccharide (LPS), which is essential to nearly all Gram-negative bacteria. LPS is transported to the outer leaflet of the OM through a yet unknown mechanism by seven proteins that comprise the LPS transport system. LptA, the only entirely periplasmic Lpt protein, bridges the periplasmic space between the IM LptB2 FGC and the OM LptDE complexes. LptA is postulated to protect the hydrophobic acyl chains of LPS as it crosses the hydrophilic periplasm, is essential to cell viability, and contains many conserved residues distributed across the protein. To identify which side chains are required for function of E. coli LptA in vivo, we performed a systematic, unbiased, high-throughput screen of the effect of 172 single alanine substitutions on cell viability utilizing an engineered BL21 derivative with a chromosomal knockout of the lptA gene. Remarkably, LptA is highly tolerant to amino acid substitution with alanine. Only four alanine mutants could not complement the chromosomal knockout; CD spectroscopy showed that these substitutions resulted in proteins with significantly altered secondary structure. In addition, 29 partial loss-of-function mutants were identified that led to OM permeability defects; interestingly, these sites were solely located within ß-strands of the central core of the protein and each resulted in misfolding of the protein. Therefore, no single residue within LptA is responsible for LPS binding, supporting previous EPR spectroscopy data indicating that sites across the entire protein work in concert to bind and transport LPS.

Exploring Primary Healthcare Experiences and Interest in Mobile Technology Engagement Amongst an Urban Population Experiencing Barriers to Care.

Mobile phone-based engagement approaches provide potential platforms for improving access to primary healthcare (PHC) services for underserved populations. We held two focus groups (February 2020) with residents (n = 25) from a low-income urban neighbourhood (downtown Vancouver, Canada), to assess recent healthcare experiences and elicit interest in mobile phone-based healthcare engagement for underserved residents. Note-based analysis, guided by interpretative description, was used to explore emerging themes. Engagement in PHC was complicated by multiple, intersecting personal-level and socio-structural factors, and experiences of stigma and discrimination from care providers. Perceived inadequacy of PHC services and pervasive discrimination reported by participants indicate a significant and ongoing need to improve client-provider relationships to address unmet health needs. Mobile phone-based engagement was endorsed, highlighting phone ownership and client-provider text-messaging, facilitated by non-clinical staff such as peers, as helpful to strengthening retention and facilitating care team connection. Concerns raised included reliability, cost, and technology and language accessibility.

Impact of the Affordable Care Act on Disparities in Access to and Outcomes After Coronary Artery Bypass Grafting.

OBJECTIVES: The aim of this study was to examine the effect of implementation of the Affordable Care Act's Medicaid expansion on access to and outcomes after coronary artery bypass grafting (CABG) surgery. METHODS: Retrospective observational study utilizing the Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (NIS) from 2011 to 2016. The southern region of the USA was used as a control and the western region as the implementation group. Univariate regression models and interrupted time series models were created to evaluate and assess the impact of the Affordable Care Act's Medicaid expansion on mortality after CABG with respect to patient race. RESULTS: From 2011 to 2016, a total of 117,819 isolated CABG operations were identified in the specified regions using the HCUP NIS. Of these, 89,918 were performed in the southern region, and the remainder were performed in the western region. The proportion of African American patients with Medicaid increased significantly in the western region after the ACA Medicaid expansion, from 13.1 to 17.6%, p = 0.034. There was no significant increase seen in the number of African American patients with Medicaid in the southern region. We found that overall, Black patients had higher mortality after CABG as compared to white patients (OR 1.15, p = 0.02); however, when broken down by region we found higher mortality among African American patients in the southern region only, with no statistically significant difference in mortality between white and Black patients in the western region. CONCLUSIONS: Implementation of the Affordable Care Act increased access to Medicaid among Black Americans but did not necessarily decrease the disparity in access to CABG or mortality after CABG between Black and white patients. When it comes to racial disparities in mortality after CABG, there are significant regional and geographic variations which have not been previously described. This finding has important implications for the development of policy and other strategies that aim to reduce these disparities.

Longitudinal profiling in patients undergoing cardiac surgery reveals postoperative changes in DNA methylation.

BACKGROUND: Cardiac surgery and cardiopulmonary bypass induce a substantial immune and inflammatory response, the overactivation of which is associated with significant pulmonary, cardiovascular, and neurologic complications. Commensurate with the immune and inflammatory response are changes in the heart and vasculature itself, which together drive postoperative complications through mechanisms that are poorly understood. Longitudinal DNA methylation profiling has the potential to identify changes in gene regulatory mechanisms that are secondary to surgery and to identify molecular processes that predict and/or cause postoperative complications. In this study, we measure DNA methylation in preoperative and postoperative whole blood samples from 96 patients undergoing cardiac surgery on cardiopulmonary bypass. RESULTS: While the vast majority of DNA methylation is unchanged by surgery after accounting for changes in cell-type composition, we identify several loci with statistically significant postoperative changes in methylation. Additionally, two of these loci are associated with new-onset postoperative atrial fibrillation, a significant complication after cardiac surgery. Paired statistical analysis, use of FACS data to support sufficient control of cell-type heterogeneity, and measurement of IL6 levels in a subset of patients add rigor to this analysis, allowing us to distinguish cell-type variability from actual changes in methylation. CONCLUSIONS: This study identifies significant changes in DNA methylation that occur immediately after cardiac surgery and demonstrates that these acute alterations in DNA methylation have the granularity to identify processes associated with major postoperative complications. This research also establishes methods for controlling for cell-type variability in a large human cohort that may be useful to deploy in other longitudinal studies of epigenetic marks in the setting of acute and chronic disease.

Quantitative Echocardiography Improves Resident Assessment of Left Ventricular Systolic Function.

Background: The use of echocardiography to assess left ventricular ejection fraction (LVEF) is an important component of anesthesiology resident education; however, there is no consensus on the most effective method for teaching this skill set. This study investigates the impact and feasibility of teaching a quantitative LVEF assessment method to anesthesiology residents, compared with teaching visual estimation techniques. Methods: We included all anesthesiology residents rotating through cardiac anesthesia at our institution from August 2020 through March 2021. Participants completed a pretest to assess baseline ability to accurately estimate LVEF. All tests consisted of transthoracic echocardiography images with standard views from 10 patients. Participants were assigned to either a control group that received teaching on visual estimation of LVEF or an intervention group that was taught quantitative LVEF assessment with the Simpson biplane method of discs. After 4 weeks, all participants were administered a postteaching exam. A retention exam was administered an additional 4 weeks later. LVEF accuracy was measured as the absolute difference between their LVEF estimation and the reference value. Results: Control and intervention groups performed similarly on the preteaching exam of LVEF estimation accuracy. Intervention-group residents demonstrated significantly improved accuracy in LVEF assessment on the postteaching exam (3.6% improvement in accuracy, confidence interval [CI], 1.23-5.97; P = .03) compared with the control group (0.60% improvement inaccuracy, CI, -1.77-2.97; P = .62). The observed improvement was not maintained through the retention exam.Conclusions: Addition of quantitative LVEF assessment to traditional teaching of visual LVEF estimation methods significantly improved the diagnostic accuracy of anesthesiology residents' left ventricular systolic function assessment.

Intravital Microscopy to Visualize Murine Small Intestinal Intraepithelial Lymphocyte Migration.

Intraepithelial lymphocytes (IELs) are critical sentinels involved in host defense and maintenance of the intestinal mucosal barrier. IELs expressing the γδ T-cell receptor provide continuous surveillance of the villous epithelium by migrating along the basement membrane and into the lateral intercellular space between adjacent enterocytes. Intravital imaging has furthered our understanding of the molecular mechanisms by which IELs navigate the epithelial compartment and interact with neighboring enterocytes at steady state and in response to infectious or inflammatory stimuli. Further, evaluating IEL migratory behavior can provide additional insight into the nature and extent of cellular interactions within the intestinal mucosa. Three protocols describe methodology to visualize small intestinal IEL motility in real time using fluorescent reporter-transgenic mice and/or fluorophore-conjugated primary antibodies and spinning-disk confocal microscopy. Using Imaris image analysis software, a fourth protocol provides a framework to analyze IEL migration and quantify lymphocyte/epithelial interactions. Together, these protocols for intravital imaging and subsequent analyses provide the basis for elucidating the spatiotemporal dynamics of mucosal immune cells and interactions with neighboring enterocytes under physiological or pathophysiological conditions. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mouse preparation and laparotomy Support Protocol: Antibody labeling of cell surface markers Basic Protocol 2: Image acquisition by spinning-disk confocal microscopy Basic Protocol 3: 4D analysis of images.

Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells.

Intestinal nematode parasites can cross the epithelial barrier, causing tissue damage and release of danger-associated molecular patterns (DAMPs) that may promote host protective type 2 immunity. We investigate whether adenosine binding to the A2B adenosine receptor (A2BAR) on intestinal epithelial cells (IECs) plays an important role. Specific blockade of IEC A2BAR inhibits the host protective memory response to the enteric helminth, Heligmosomoides polygyrus bakeri (Hpb), including disruption of granuloma development at the host-parasite interface. Memory T cell development is blocked during the primary response, and transcriptional analyses reveal profound impairment of IEC activation. Extracellular ATP is visualized 24 h after inoculation and is shown in CD39-deficient mice to be critical for the adenosine production mediating the initiation of type 2 immunity. Our studies indicate a potent adenosine-mediated IEC pathway that, along with the tuft cell circuit, is critical for the activation of type 2 immunity.

DNA Methylation-Based Prediction of Post-operative Atrial Fibrillation.

Background: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and post-operative atrial fibrillation (POAF) is a major healthcare burden, contributing to an increased risk of stroke, kidney failure, heart attack and death. Genetic studies have identified associations with AF, but no molecular diagnostic exists to predict POAF based on pre-operative measurements. Such a tool would be of great value for perioperative planning to improve patient care and reduce healthcare costs. In this pilot study of epigenetic precision medicine in the perioperative period, we carried out bisulfite sequencing to measure DNA methylation status in blood collected from patients prior to cardiac surgery to identify biosignatures of POAF. Methods: We enrolled 221 patients undergoing cardiac surgery in this prospective observational study. DNA methylation measurements were obtained from blood samples drawn from awake patients prior to surgery. After controlling for clinical and methylation covariates, we analyzed DNA methylation loci in the discovery cohort of 110 patients for association with POAF. We also constructed predictive models for POAF using clinical and DNA methylation data. We subsequently performed targeted analyses of a separate cohort of 101 cardiac surgical patients to measure the methylation status solely of significant methylation loci in the discovery cohort. Results: A total of 47 patients in the discovery cohort (42.7%) and 43 patients in the validation cohort (42.6%) developed POAF. We identified 12 CpGs that were statistically significant in the discovery cohort after correcting for multiple hypothesis testing. Of these sites, 6 were amenable to targeted bisulfite sequencing and chr16:24640902 was statistically significant in the validation cohort. In addition, the methylation POAF prediction model had an AUC of 0.79 in the validation cohort. Conclusions: We have identified DNA methylation biomarkers that can predict future occurrence of POAF associated with cardiac surgery. This research demonstrates the use of precision medicine to develop models combining epigenomic and clinical data to predict disease.