Structural maturation of myofilaments in engineered 3D cardiac microtissues characterized using small angle x-ray scattering.

Understanding the structural and functional development of human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) is essential to engineering cardiac tissue that enables pharmaceutical testing, modeling diseases, and designing therapies. Here we use a method not commonly applied to biological materials, small angle x-ray scattering, to characterize the structural development of hiPSC-CMs within three-dimensional engineered tissues during their preliminary stages of maturation. An x-ray scattering experimental method enables the reliable characterization of the cardiomyocyte myofilament spacing with maturation time. The myofilament lattice spacing monotonically decreases as the tissue matures from its initial post-seeding state over the span of 10 days. Visualization of the spacing at a grid of positions in the tissue provides an approach to characterizing the maturation and organization of cardiomyocyte myofilaments and has the potential to help elucidate mechanisms of pathophysiology, and disease progression, thereby stimulating new biological hypotheses in stem cell engineering.

Surgical Approach and Dislocation Risk After Hemiarthroplasty in Geriatric Patients With Femoral Neck Fracture With and Without Cognitive Impairments-Does Cognitive Impairment Influence Dislocation Risk?

OBJECTIVES: To determine whether there is an association between surgical approach and dislocation risk in patients with cognitive impairment compared with those without cognitive impairment treated with hemiarthroplasty for femoral neck fracture. DESIGN: Retrospective study. SETTING: Large, multicenter health system. PATIENTS/PARTICIPANTS: One thousand four hundred eighty-one patients who underwent hemiarthroplasty for femoral neck fractures. 828 hips met inclusion criteria, 290 (35.0%) were cognitively impaired, and 538 (65.0%) were cognitively intact. INTERVENTION: Hemiarthroplasty. MAIN OUTCOME MEASURE: Prosthetic hip dislocation. RESULTS: The overall dislocation rate was 2.1% (17 of 828), 3.4% (10 of 290) in the cognitively impaired group, and 1.3% (7 of 538) in the cognitively intact group with a median time to dislocation of 20.5 days (range 2-326 days), 24.5 days (range 3-326 days), and 19.0 days (range 2-36 days), respectively. In the entire cohort, there were no dislocations (0 of 58) with the direct anterior approach (DA); 1.1% (6 of 553) and 5.1% (11 of 217) dislocated with the modified Hardinge (MH) and posterior approaches (PA), respectively. In the cognitively impaired group, there were no dislocations with the DA (0 of 19); 1.5% (3 of 202) and 10.1% (7 of 69) dislocated with the MH and PA, respectively. In the cognitively intact group, there were no dislocations (0 of 39) with the DA; 0.85% (3 of 351) and 2.7% (4 of 148) dislocated with the MH and PA, respectively. There were statistically significant associations between surgical approach and dislocation in the entire cohort and the cognitively impaired group when comparing the MH and PA groups. This was not observed in the cognitively intact group. Patients who dislocated had 3.2 times (95% CI 1.2, 8.7) ( P = 0.0226) the hazard of death compared with patients who did not dislocate. Dislocation effectively increased the risk of death by 221% (HR 3.2 95% CI 1.2, 8.7) ( P = 0.0226). CONCLUSIONS: In this patient population, the PA has a higher dislocation rate than other approaches and has an especially high rate of dislocation when the patients were cognitively impaired. The authors of this study suggest careful consideration of surgical approach when treating these injuries. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Establishing a balloon pulmonary angioplasty program for chronic thromboembolic pulmonary hypertension: A United States single-center experience.

INTRODUCTION: Balloon pulmonary angioplasty (BPA) is a less invasive treatment alternative for patients with chronic thromboembolic pulmonary hypertension (CTEPH) who are unable to move forward with pulmonary thromboendarterectomy. This report describes a single-center experience with a nascent BPA program in the United States (US). METHODS: All patients who underwent BPA between August 2018-2021 were included in this retrospective, single-center observational cohort. Pre- and post-procedure clinical information was collected, along with procedural characteristics. RESULTS: Thirty patients began their BPA series during the study period. The majority of patients had segmental disease (n = 25, 83.3%). A total of 135 BPA procedures were performed on 417 segments. On average, patients completed 4.5 sessions and the majority of patients (n = 23, 76.7%) underwent more than 2. There were 24 episodes of hemoptysis and 20 procedural events that required treatment, typically with either heparin reversal or balloon tamponade. Of 26 participants with completed series, mean PA pressure (-6 mmHg, 95% CI -9 to -4 mmHg, p = 0.0001), PVR (-1.9 Wood units, 95% CI -2.9 to -1.0, p = 0.0002), and pulmonary compliance (-1.0 mL/mmHg, 95% CI -1.5 to -0.5, p = 0.0002) improved. Improvement was also seen in NYHA functional classification and walk distance (p = 0.01). Two deaths occurred, with one death peri-procedurally. CONCLUSION: This paper describes an early experience with BPA at a single US center. Improvement in non-invasive and invasive metrics were seen without adding a significant morbidity to an already high-risk patient population.

XP-endo Finisher effectively reduces hard-tissue debris accumulated in root canals with isthmus after preparation with a reciprocating file system.

This study compared the accumulated hard-tissue debris (AHTD) after preparation with WaveOne Gold (WOG) to XP-endo Shaper (XPS), without and with a supplementary step using XP-endo Finisher (XPF) using clinically applicable irrigation. Twenty-four mesial roots with two canals and single foramen were micro-CT-scanned and matched. Scans were also taken after preparation with WOG or XPS, and after XPF. Irrigation with 2.5% NaOCl (total: 17 ml per canal) and 17% EDTA (2.5 ml per canal) was performed using a 30ga Max-I-Probe needle placed up to the working length. Morphological parameters were calculated and compared within and among groups. XPF significantly reduced unprepared area within XPS and WOG groups, and AHTD within WOG (p < 0.05). There were no significant differences between WOG and XPS after preparation and after XPF (p > 0.05). In conclusion, WOG and XPS produced a similar volume of AHTD, but the supplementary step with XPF decreased the AHTD in the WOG group.

Graphia: A platform for the graph-based visualisation and analysis of high dimensional data.

Graphia is an open-source platform created for the graph-based analysis of the huge amounts of quantitative and qualitative data currently being generated from the study of genomes, genes, proteins metabolites and cells. Core to Graphia's functionality is support for the calculation of correlation matrices from any tabular matrix of continuous or discrete values, whereupon the software is designed to rapidly visualise the often very large graphs that result in 2D or 3D space. Following graph construction, an extensive range of measurement algorithms, routines for graph transformation, and options for the visualisation of node and edge attributes are available, for graph exploration and analysis. Combined, these provide a powerful solution for the interpretation of high-dimensional data from many sources, or data already in the form of a network or equivalent adjacency matrix. Several use cases of Graphia are described, to showcase its wide range of applications in the analysis biological data. Graphia runs on all major desktop operating systems, is extensible through the deployment of plugins and is freely available to download from https://graphia.app/.

Transferable Global Rating Scales in the Validation of the ArthroSim™ Virtual Reality Arthroscopy Simulator.

INTRODUCTION: Virtual reality arthroscopic simulators are becoming increasingly prevalent in the orthopaedic training environment. The construct validity of the ArthroSim virtual reality simulator (TolTech Touch of Life Technologies, Aurora, Colorado) has been established based on time to completion comparison between candidates of differing levels of surgical experience. This study aims to establish the construct validity of the ArthroSim virtual reality simulator using validated global rating scales that allow direct comparison with intraoperative performance. MATERIALS AND METHODS: Eight novices (medical students), eight intermediates (registrars), and seven experts (consultants) were assessed using the Imperial Global Arthroscopy Rating Scale (IGARS) and the Arthroscopic Surgical Skills Evaluation Tool (ASSET) scoring systems while carrying out a standardised basic diagnostic knee arthroscopy using linked and anonymised recordings of both the arthroscopy video output and candidate's hand posture and position. Time to completion was recorded and the expert group also filled out questionnaires assessing the face and content validity of the simulator. RESULTS: The mean IGARS/ASSET scores for the novice, intermediate and expert groups were 14/11, 29/22, and 46/36 respectively. The difference in score between each of the groups was statistically significant (p<0.05). The average time to completion was 257 seconds, 305 seconds, and 204 seconds respectively. The time to completion was not significantly different between the groups (p=0.6). CONCLUSIONS: The ArthroSim virtual reality simulator could effectively distinguish between candidates of differing experience levels using validated global rating scales and therefore demonstrated construct validity.

Buprenorphine management: a conundrum for the anesthesiologist and beyond - a one-act play.

We have witnessed a worldwide upsurge of streamlined enhanced recovery after surgery (ERAS) pathways advocating for consistency and compliance within their guidelines. At a recent national conference, two experts defended their institutional policies on perioperative management of buprenorphine, one defending its continuation, while the other suggesting its discontinuation. The moderator diplomatically proclaimed the need to have guidance at the institutional level and following it for favorable patient outcomes. Unfortunately, perioperative management of buprenorphine remains an understudied topic with a lack of national guidelines leading to variations at a local level despite its increased use nationally in the current opioid crisis. Although the moderator made a valid statement, we demonstrate via our one-act play the importance of recognizing a subset of the population within an ERAS pathway that necessitates multidisciplinary discussion, communication, and patient-centric care to formulate a perioperative plan coordinating a patient's care. More robust research is needed to minimize variability in current practices and to further develop comprehensive evidence-based guidelines that encompass risk factors and anticipated postsurgical and peripartum pain for patients on buprenorphine.

Treatment Trajectories During and After a Medication Trial for Opioid Use Disorder: Moving from Research as Usual to Treatment as Usual.

OBJECTIVES: The effectiveness of treatment incorporating relapse prevention medications for opioid use disorder (OUD) is typically examined in research using rigidly predefined endpoints of success versus failure, usually over a single episode of care. But this perspective may not adequately portray the nonlinear trajectories typical of real-world treatment courses in this chronic, remitting, and relapsing disorder. METHODS: This descriptive study examined 12-month treatment trajectories of n = 60 patients enrolled at a single site of a larger multisite randomized controlled trial examining the comparative effectiveness of buprenorphine versus extended-release naltrexone. While the parent study provided medication treatment through the research protocol for 6 months, this study documents treatment up to 12 months, including medications, provided through standard community resources (treatment as usual) outside of the protocol. RESULTS: Some patients continued medications past the end of the study intervention, whereas others did not. Some patients initiated medications other than the one assigned by the study. Some patients switched from 1 medication to the other. Many patients returned to treatment after 1 or more periods of dropout and/or relapse. Patients utilized multiple episodes of bed-based care, including short-term acute residential and long-term residential treatment, and also recovery housing supports. Described trajectories are also depicted graphically. At 12 months, while rates of continuous treatment retention were low (8%), rates of cross-sectional treatment engagement including return to treatment after drop out were higher (35%). CONCLUSIONS: This description of nonlinear treatment trajectories highlights the potential benefits of flexibility and optimism in the promotion of re-engagement, despite interim outcomes that might traditionally be considered "failure" endpoints.

Patient Outcomes and Cost-Effectiveness of a Sepsis Care Quality Improvement Program in a Health System.

OBJECTIVES: Assess patient outcomes in patients with suspected infection and the cost-effectiveness of implementing a quality improvement program. DESIGN, SETTING, AND PARTICIPANTS: We conducted an observational single-center study of 13,877 adults with suspected infection between March 1, 2014, and July 31, 2017. The 18-month period before and after the effective date for mandated reporting of the sepsis bundle was examined. The Sequential Organ Failure Assessment score and culture and antibiotic orders were used to identify patients meeting Sepsis-3 criteria from the electronic health record. INTERVENTIONS: The following interventions were performed as follows: 1) multidisciplinary sepsis committee with sepsis coordinator and data abstractor; 2) education campaign; 3) electronic health record tools; and 4) a Modified Early Warning System. MAIN OUTCOMES AND MEASURES: Primary health outcomes were in-hospital death and length of stay. The incremental cost-effectiveness ratio was calculated and the empirical 95% CI for the incremental cost-effectiveness ratio was estimated from 5,000 bootstrap samples. RESULTS: In multivariable analysis, the odds ratio for in-hospital death in the post- versus pre-implementation periods was 0.70 (95% CI, 0.57-0.86) in those with suspected infection, and the hazard ratio for time to discharge was 1.25 (95% CI, 1.20-1.29). Similarly, a decrease in the odds for in-hospital death and an increase in the speed to discharge was observed for the subset that met Sepsis-3 criteria. The program was cost saving in patients with suspected infection (-$272,645.7; 95% CI, -$757,970.3 to -$79,667.7). Cost savings were also observed in the Sepsis-3 group. CONCLUSIONS AND RELEVANCE: Our health system's program designed to adhere to the sepsis bundle metrics led to decreased mortality and length of stay in a cost-effective manner in a much larger catchment than just the cohort meeting the Centers for Medicare and Medicaid Services measures. Our single-center model of interventions may serve as a practice-based benchmark for hospitalized patients with suspected infection.

Genetic, Inflammatory, and Epithelial Cell Differentiation Factors Control Expression of Human Calpain-14.

Eosinophilic esophagitis (EoE) is a chronic, food-driven allergic disease resulting in eosinophilic esophageal inflammation. We recently found that EoE susceptibility is associated with genetic variants in the promoter of CAPN14, a gene with reported esophagus-specific expression. CAPN14 is dynamically up-regulated as a function of EoE disease activity and after exposure of epithelial cells to interleukin-13 (IL-13). Herein, we aimed to explore molecular modulation of CAPN14 expression. We identified three putative binding sites for the IL-13-activated transcription factor STAT6 in the promoter and first intron of CAPN14 Luciferase reporter assays revealed that the two most distal STAT6 elements were required for the ∼10-fold increase in promoter activity subsequent to stimulation with IL-13 or IL-4, and also for the genotype-dependent reduction in IL-13-induced promoter activity. One of the STAT6 elements in the promoter was necessary for IL-13-mediated induction of CAPN14 promoter activity while the other STAT6 promoter element was necessary for full induction. Chromatin immunoprecipitation in IL-13 stimulated esophageal epithelial cells was used to further support STAT6 binding to the promoter of CAPN14 at these STAT6 binding sites. The highest CAPN14 and calpain-14 expression occurred with IL-13 or IL-4 stimulation of esophageal epithelial cells under culture conditions that allow the cells to differentiate into a stratified epithelium. This work corroborates a candidate molecular mechanism for EoE disease etiology in which the risk variant at 2p23 dampens CAPN14 expression in differentiated esophageal epithelial cells following IL-13/STAT6 induction of CAPN14 promoter activity.

Expression of the HSF4 DNA binding domain-EGFP hybrid gene recreates early childhood lamellar cataract in transgenic mice.

PURPOSE: The clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice. METHODS: Mutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4. RESULTS: We recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation. CONCLUSIONS: Recreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.

Cell-type-dependent access of HSF1 and HSF4 to αB-crystallin promoter during heat shock.

Epithelial cells and fibroblasts both express heat shock transcription factors, HSF1 and HSF4, yet they respond to heat shock differentially. For example, while HSP70 is induced in both cell types, the small heat shock protein, αB-crystallin gene (CRYAB) that contains a canonical heat shock promoter, is only induced in fibroblasts. A canonical heat shock promoter contains three or more inverted repeats of the pentanucleotide 5'-nGAAn-3' that make the heat shock element. It is known that, in vitro, promoter architecture (the order and spacing of these repeats) impacts the interaction of various heat shock transcription factors (HSFs) with the heat shock promoter, but in vivo relevance of these binding preferences so far as the expression is concerned is poorly understood. In this report, we first establish cell-type-dependent differential expression of CRYAB in four established cell lines and then working with adult human retinal pigment epithelial cells and NIH3T3 fibroblasts and employing chromatin immunoprecipitation, attempt to relate expression to promoter occupancy by HSF1 and HSF4. We show that HSF4 occupies only CRYAB and not HSP70 promoter in epithelial cells, while HSF1 occupies only HSP70 promoter in both cell types, and cryab promoter, only in heat shocked fibroblasts; HSF4, on the other hand, is never seen on these two promoters in NIH3T3 fibroblasts. This comparative analysis with CRYAB and HSP70 demonstrates that differential heat shock response is controlled by cell-type-dependent access of HSFs (HSF1 and HSF4) to specific promoters, independent of the promoter architecture.

Hypoxia-inducible factor signaling provides protection in Clostridium difficile-induced intestinal injury.

BACKGROUND & AIMS: Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Antibiotic resistance and increased virulence of strains have increased the number of C difficile-related deaths worldwide. The innate host response mechanisms to C difficile are not resolved; we propose that hypoxia-inducible factor (HIF-1) has an innate, protective role in C difficile colitis. We studied the impact of C difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1alpha in C difficile-mediated injury/inflammation. METHODS: We assessed HIF-1alpha mRNA and protein levels and DNA binding in human mucosal biopsy samples and Caco-2 cells following exposure to C difficile toxins. We used the mouse ileal loop model of C difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1alpha in the intestinal epithelium were used to assess the effects of HIF-1alpha signaling in response to C difficile toxin. RESULTS: Mucosal biopsy specimens and Caco-2 cells exposed to C difficile toxin had a significant increase in HIF-1alpha transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1alpha accumulation was attenuated by nitric oxide synthase inhibitors. In vivo deletion of intestinal epithelial HIF-1alpha resulted in more severe, toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1alpha with dimethyloxallyl glycine attenuated toxin-induced injury and inflammation. This was associated with induction of HIF-1-regulated protective factors (such as vascular endothelial growth factor-alpha, CD73, and intestinal trefoil factor) and down-regulation of proinflammatory molecules such as tumor necrosis factor and Cxcl1. CONCLUSIONS: HIF-1alpha protects the intestinal mucosa from C difficile toxins. The innate protective actions of HIF-1alpha in response to C difficile toxins be developed as therapeutics for C difficile-associated disease.