Increased Activity of Epithelial Cdc42 Rho GTPase and Tight Junction Permeability in the Cftr Knockout Intestine.

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/ß-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pull-down assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled 2. Using a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3kD dextran relative to WT. In Cftr KO relative to WT enteroids, leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/ß-catenin signaling with Endo-IWR1. Increased proliferation or inhibition of Cdc42 activity with ML141 had no effect on WT enteroid permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3kD dextran and tight-junction impermeant 500 kD dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.

High-Fidelity Bleeding Control Simulation Scenario During Medical Student Orientation Improves Students' Self-Reported Ability to Identify and Treat Life-Threatening Bleeding with 3-year Follow-up.

OBJECTIVE: Increasingly, medical schools integrate clinical skills into early didactic coursework. The Stop the Bleed® Campaign emphasizes prehospital hemorrhage control to reduce preventable deaths; however, this course overlooks team interactions. We assessed the impact of high-fidelity simulation during medical student orientation on identification and treatment of life-threatening hemorrhage in a team setting. DESIGN: In this mixed method, prospective pre-, post-, and follow-up survey analysis assessing student knowledge and attitudes, student teams encountered a standardized patient in a prehospital environment with pulsatile bleeding from an extremity wound. Individual students completed surveys assessing previous experience, willingness and ability to assist bleeding person(s), and knowledge and attitudes about tourniquets. Postscenario, faculty preceptors made qualitative observations on teamwork. SETTING: Medical student orientation at a tertiary care academic medical center with long-term follow-up. PARTICIPANTS: Medical students (N = 150). RESULTS: Ninety students (60%) completed both pre- and postsimulation questionnaires. Sixteen (17%) students had previous tourniquet training experience although none had applied a tourniquet outside of training. Postsimulation, students reported increased likelihood of providing treatment until additional help arrived (p = 0.035), improved ability to identify life-threatening hemorrhage (p < 0.001), and more favorable opinions about tourniquet use (p < 0.001) and potential for limb-salvage (p = 0.018). Long-term follow-up respondents (n = 34, 23%) reported increased ability to identify life-threatening hemorrhage (p = 0.010) and universal willingness to intervene until additional help arrived. Follow-up survey responses elicited themes in hemorrhage control including recognition of the importance of continuous pressure, appropriate use of tourniquets, a desire for repeated team training, and the recognition of clerkship rotations as an optimal setting for skill reinforcement. Preceptors noted variable team responses but uniformly endorsed the exercise. CONCLUSIONS: High-fidelity bleeding simulation during medical student orientation improved students' knowledge and attitudes about treating life-threatening hemorrhage and served as an introduction to team-based emergency care. Future studies should further explore team training and hemorrhage control education.

Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of CFTR in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR was compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR was quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared to control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR was accompanied by decreases in mRNA for the Cl-/HCO3- exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1) which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low risk, high risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by underlying failure of CFTR function.

Examining the Aftermath of Virtual Interviewing on Surgical Residency Matches.

INTRODUCTION: Residency interviews have traditionally been conducted in person; however, COVID-19 forced programs to shift to virtual interviewing. This study delineated the nationwide trends observed after virtual interviewing across multiple application cycles on both surgical residency applicant competitiveness and program workload. METHODS: Publicly available National Residency Matching Program applicant and program data were retrospectively reviewed. Applicant competitiveness was assessed using a validated competitive index (# positions ranked/match rate). Interview types included in-person (2010-2020) or virtual (2021-2023), and programs were classified as general surgery (GS), surgical subspecialty (SS) - orthopedics, otolaryngology and neurosurgery, and integrated specialty (IS) - plastic, thoracic, and vascular surgery. RESULTS: When comparing in-person to virtual cohorts, the competitive index has increased in GS (0.97 ± 0.00 to 1.05 ± 0.01, P < 0.001), SS (0.97 ± 0.02 to 1.06 ± 0.01 P < 0.001), and IS (0.93 ± 0.06 to 1.12 ± 0.03, P = 0.001). United Sates Medical Licensing Examination Step scores and research experiences increased over time in GS and SS (P < 0.05). Program workload, represented by number of applications received per program increased in GS, IS, and SS (P < 0.05), as well as the number of interviews conducted in GS and SS (P < 0.05). Importantly, match rate remained stable in GS and IS, with a decrease in SS (0.69 ± 0.03 to 0.63 ± 0.02, P = 0.04). CONCLUSIONS: The residency application process has been irrevocably changed due to COVID-19. The rise in applicant volume and competitiveness places unique strains on applicants and programs. Additional modifications such as signaling and ACGME guidance are needed to help alleviate strain and ensure that residents and programs alike find their best fit.

Identifying and Reducing Insulin Errors in the Simulated Military Critical Care Air Transport Environment: A Human Factors Approach.

INTRODUCTION: During high-fidelity simulations in the Critical Care Air Transport (CCAT) Advanced course, we identified a high frequency of insulin medication errors and sought strategies to reduce them using a human factors approach. MATERIALS AND METHODS: Of 169 eligible CCAT simulations, 22 were randomly selected for retrospective audio-video review to establish a baseline frequency of insulin medication errors. Using the Human Factors Analysis Classification System, dosing errors, defined as a physician ordering an inappropriate dose, were categorized as decision-based; administration errors, defined as a clinician preparing and administering a dose different than ordered, were categorized as skill-based. Next, 3 a priori interventions were developed to decrease the frequency of insulin medication errors, and these were grouped into 2 study arms. Arm 1 included a didactic session reviewing a sliding-scale insulin (SSI) dosing protocol and a hands-on exercise requiring all CCAT teams to practice preparing 10 units of insulin including a 2-person check. Arm 2 contained arm 1 interventions and added an SSI cognitive aid available to students during simulation. Frequency and type of insulin medication errors were collected for both arms with 93 simulations for arm 1 (January-August 2021) and 139 for arm 2 (August 2021-July 2022). The frequency of decision-based and skill-based errors was compared across control and intervention arms. RESULTS: Baseline insulin medication error rates were as follows: decision-based error occurred in 6/22 (27.3%) simulations and skill-based error occurred in 6/22 (27.3%). Five of the 6 skill-based errors resulted in administration of a 10-fold higher dose than ordered. The post-intervention decision-based error rates were 9/93 (9.7%) and 23/139 (2.2%), respectively, for arms 1 and 2. Compared to baseline error rates, both arm 1 (P = .04) and arm 2 (P < .001) had a significantly lower rate of decision-based errors. Additionally, arm 2 had a significantly lower decision-based error rate compared to arm 1 (P = .015). For skill-based preparation errors, 1/93 (1.1%) occurred in arm 1 and 4/139 (2.9%) occurred in arm 2. Compared to baseline, this represents a significant decrease in skill-based error in both arm 1 (P < .001) and arm 2 (P < .001). There were no significant differences in skill-based error between arms 1 and 2. CONCLUSIONS: This study demonstrates the value of descriptive error analysis during high-fidelity simulation using audio-video review and effective risk mitigation using training and cognitive aids to reduce medication errors in CCAT. As demonstrated by post-intervention observations, a human factors approach successfully reduced decision-based error by using didactic training and cognitive aids and reduced skill-based error using hands-on training. We recommend the development of a Clinical Practice Guideline including an SSI protocol, guidelines for a 2-person check, and a cognitive aid for implementation with deployed CCAT teams. Furthermore, hands-on training for insulin preparation and administration should be incorporated into home station sustainment training to reduced medication errors in the operational environment.

Fecal dysbiosis and inflammation in intestinal-specific Cftr knockout mice on regimens preventing intestinal obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of cystic fibrosis (CF), which may be refractory to ion channel CF transmembrane conductance regulator (CFTR) modulator therapy. People with CF exhibit intestinal dysbiosis, which has the potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia, leukocytes, and other tissues. Here, we investigate the contribution of intestinal epithelium-specific loss of Cftr [iCftr knockout (KO)] to dysbiosis and inflammation in mice treated with either of two antiobstructive dietary regimens necessary to maintain CF mouse models [polyethylene glycol (PEG) laxative or a liquid diet (LiqD)]. Feces collected from iCftr KO mice and their wild-type (WT) sex-matched littermates were used to measure fecal calprotectin to evaluate inflammation and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT mice that consumed either PEG or LiqD. PEG iCftr KO mice did not show a change in α diversity versus WT mice but demonstrated a significant difference in microbial composition (ß diversity) with included increases in the phylum Proteobacteria, the family Peptostreptococcaceae, four genera of Clostridia including C. innocuum, and the mucolytic genus Akkermansia. Fecal microbiome analysis of LiqD-fed iCftr KO mice showed both decreased α diversity and differences in microbial composition with increases in the Proteobacteria family Enterobacteriaceae, Firmicutes families Clostridiaceae and Peptostreptococcaceae, and enrichment of Clostridium perfringens, C. innocuum, C. difficile, mucolytic Ruminococcus gnavus, and reduction of Akkermansia. It was concluded that epithelium-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of pan Cftr KO mice.NEW & NOTEWORTHY Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CF transmembrane conductance regulator (CFTR) that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR [inducible Cftr knockout (KO)] in mice is sufficient to induce intestinal dysbiosis and inflammation. Experiments were performed on mice consuming two dietary regimens routinely used to prevent obstruction in CF mice.

Fecal Dysbiosis and Inflammation in Intestinal-Specific Cftr Knockout Mice on Regimens Preventing Intestinal Obstruction.

Chronic intestinal inflammation is a poorly understood manifestation of Cystic Fibrosis (CF), which may be refractory to ion channel CFTR modulator therapy. People with CF exhibit intestinal dysbiosis which has potential for stimulating intestinal and systemic inflammation. CFTR is expressed in organ epithelia and in the leukocyte population. Here, we investigate the contribution of intestinal epithelial-specific loss of Cftr (iCftr KO) to dysbiosis and inflammation in mice treated with either of two anti-obstructive dietary regimens necessary to maintain CF mouse models (PEG laxative or a liquid diet, LiqD). Feces collected from iCftr KO mice and their wildtype (WT) sex-matched littermates were used to measure fecal calprotectin and to perform 16S rRNA sequencing to characterize the gut microbiome. Fecal calprotectin was elevated in iCftr KO relative to WT samples of mice consuming either PEG or LiqD. PEG iCftr KO mice did not show a change in α-diversity versus WT but demonstrated a significant difference in microbial composition (ß-diversity) with increases in phylum Proteobacteria , family Peptostreptococcaceae , four genera of Clostridia including C. innocuum , and mucolytic genus Akkermansia . Fecal microbiome analysis of LiqD iCftr KO mice showed both decreased α-diversity and differences in microbial composition with increases in Proteobacteria family Enterobacteriaceae , Firmicutes families Clostridiaceae and Peptostreptococcaceae , and enrichment of Clostridium perfringens , C. innocuum , C. difficile , mucolytic Ruminococcus gnavus , and reduction of Akkermansia . It was concluded that epithelial-specific loss of Cftr is a major driver of CF intestinal dysbiosis and inflammation with significant similarities to previous studies of global Cftr KO mice. New and noteworthy: Chronic intestinal inflammation is a manifestation of cystic fibrosis (CF), a disease caused by loss of the anion channel CFTR that is expressed in many tissues. This study shows that intestinal epithelial cell-specific loss of CFTR (iCftr KO) in mice is sufficient to induce intestinal dysbiosis and inflammation. Studies were performed on mice consuming either dietary regimen (PEG laxative or liquid diet) routinely used to prevent obstruction in CF mice.

Impact evaluation of the family expectations program and moderation by sociodemographic disadvantage.

The federal government, through the Administration for Children and Families (ACF), has funded community-based relationship education programs for couples, individuals, and families, with a strong focus on serving economically disadvantaged and racially diverse families. This study evaluated the impact of a 36-hour, workshop-based couple relationship education program that was funded by ACF using a randomized controlled trial (RCT) design and intent-to-treat (ITT) analyses. Participants were 1320 couples who were either expecting a baby or had a baby within the past 3 months, at the time of enrollment. Follow-up surveys were administered 12 months later. Analyses evaluated program impacts on relationship stability, constructive communication, and destructive conflict compared to a no-treatment control group. Analyses showed a statistically significant impact of the program on destructive conflict (d = 0.10) but not on constructive communication (d = 0.06) or stability (dCox  = 0.10). Based on findings from previous evaluations, we also examined whether participants' levels of sociodemographic disadvantage moderated these effects. There was significant moderation by sociodemographic disadvantage on constructive communication and destructive conflict, but not on stability. Effects were observed for those at higher levels of sociodemographic disadvantage.

Synthesis and Evaluation of Pseudomonas aeruginosa ATP Synthase Inhibitors.

New antibiotics with unique biological targets are desperately needed to combat the growing number of resistant bacterial pathogens. ATP synthase, a critical protein found in all life, has recently become a target of interest for antibiotic development due to the success of the anti-tuberculosis drug bedaquiline, and while many groups have worked on developing drugs to target bacterial ATP synthase, few have been successful at inhibiting Pseudomonas aeruginosa (PA) ATP synthase specifically. PA is one of the leading causes of resistant nosocomial infections across the world and is extremely challenging to treat due to its various antibiotic resistance mechanisms for most commonly used antibiotics. Herein, we detail the synthesis and evaluation of a series of C1/C2 quinoline analogues for their ability to inhibit PA ATP synthase and act as antibiotics against wild-type PA. From this survey, we found six compounds capable of inhibiting PA ATP synthase in vitro showing that bulky/hydrophobic C1/C2 substitutions are preferred. The strongest inhibitor showed an IC50 of 10 µg/mL and decreased activity of PA ATP synthase to 24% relative to the control. While none of the compounds were able to inhibit wild-type PA in cell culture, two showed improved inhibition of PA growth when permeability of the outer membrane was increased or efflux was knocked out, thus demonstrating that these compounds could be further developed into efficacious antibiotics.

Laparoscopic Appendectomy and Cholecystectomy Timing Predicts Hand-Offs but Not Miscounts.

INTRODUCTION: Intraoperative hand-offs are poorly coordinated and associated with risk of surgical miscount. We evaluated hand-off patterns for nursing staff during two common operations hypothesizing that hand-off patterns would be associated with increased surgical miscounts and vary during operations performed standard versus nonstandard operating hours. METHODS: We retrospectively analyzed laparoscopic cholecystectomy (N = 3888) and appendectomy (N = 1768) from 2012 to 2021 at a single institution using electronic medical records. We evaluated intraoperative hand-off patterns and the presence of miscounts for operations performed during standard versus nonstandard hours. Standard operating hours were defined as M-F 7:30 am to 5:00 pm. RESULTS: Across 5656 operations, 10 cases had surgical miscounts and were significantly longer than those without (156.5 versus 101 min P = 0.0178). More than half (51.3%) of cases had no identified hand-offs, and 42.9% of cases occurred during nonstandard hours. Cases during standard versus nonstandard hours were more likely to have hand-offs (56.0% versus 38.9%), P < 0.0001 and had shorter interval between hand-offs (64 versus 75 min), P < 0.0001. The period between patient entry to the room and intubation, which includes initial counts, had a disproportionately high percentage of hand-offs (P < 0.0001). CONCLUSIONS: Variability in hand-off occurrence and frequency in operations performed during standard and nonstandard hours suggest that hand-offs are influenced by staffing patterns. Few surgical miscounts occurred but were associated with longer cases. Hand-offs disproportionately occurred between patient entry and intubation, with a potential for disruption of initial instrument counts. Future work optimizing hand-off coordination is an opportunity to mitigate risk to patients.

Goblet cell hyperplasia is not epithelial-autonomous in the Cftr knockout intestine.

Goblet cell hyperplasia is an important manifestation of cystic fibrosis (CF) disease in epithelial-lined organs. Explants of CF airway epithelium show normalization of goblet cell numbers; therefore, we hypothesized that small intestinal enteroids from Cftr knockout (KO) mice would not exhibit goblet cell hyperplasia. Toll-like receptors 2 and 4 (Tlr2 and Tlr4) were investigated as markers of inflammation and influence on goblet cell differentiation. Ex vivo studies found goblet cell hyperplasia in Cftr KO jejunum compared with wild-type (WT) mice. IL-13, SAM pointed domain-containing ETS transcription factor (Spdef), Tlr2, and Tlr4 protein expression were increased in Cftr KO intestine relative to WT. In contrast, WT and Cftr KO enteroids did not exhibit differences in basal or IL-13-stimulated goblet cell numbers, or differences in expression of Tlr2, Tlr4, and Spdef. Ileal goblet cell numbers in Cftr KO/Tlr4 KO and Cftr KO/Tlr2 KO mice were not different from Cftr KO mice, but enumeration was confounded by altered mucosal morphology. Treatment with Tlr4 agonist LPS did not affect goblet cell numbers in WT or Cftr KO enteroids, whereas the Tlr2 agonist Pam3Csk4 stimulated goblet cell hyperplasia in both genotypes. Pam3Csk4 stimulation of goblet cell numbers was associated with suppression of Notch1 and Neurog3 expression and upregulated determinants of goblet cell differentiation. We conclude that goblet cell hyperplasia and inflammation of the Cftr KO small intestine are not exhibited by enteroids, indicating that this manifestation of CF intestinal disease is not epithelial-automatous but secondary to the altered CF intestinal environment.NEW & NOTEWORTHY Studies of small intestinal organoids from cystic fibrosis (CF) mice show that goblet cell hyperplasia and increased Toll-like receptor 2/4 expression are not primary manifestations of the CF intestine. Intestinal goblet cell hyperplasia in the CF mice was not strongly altered by genetic ablation of Tlr2 and Tlr 4, but could be induced in both wild-type and CF intestinal organoids by a Tlr2-dependent suppression of Notch signaling.

Modeling Performance of Open Surgical Cases.

INTRODUCTION: Previous efforts used digital video to develop computer-generated assessments of surgical hand motion economy and fluidity of motion. This study tests how well previously trained assessment models match expert ratings of suturing and tying video clips recorded in a new operating room (OR) setting. METHODS: Enabled through computer vision of the hands, this study tests the applicability of assessments born out of benchtop simulations to in vivo suturing and tying tasks recorded in the OR. RESULTS: Compared with expert ratings, computer-generated assessments for fluidity of motion (slope = 0.83, intercept = 1.77, R2 = 0.55) performed better than motion economy (slope = 0.73, intercept = 2.04, R2 = 0.49), although 85% of ratings for both models were within ±2 of the expert response. Neither assessment performed as well in the OR as they did on the training data. Assessments were sensitive to changing hand postures, dropped ligatures, and poor tissue contact-features typically missing from training data. Computer-generated assessment of OR tasks was contingent on a clear, consistent view of both surgeon's hands. CONCLUSIONS: Computer-generated assessment may help provide formative feedback during deliberate practice, albeit with greater variability in the OR compared with benchtop simulations. Future work will benefit from expanded available bimanual video records.

Zn-based physiometacomposite nanoparticles: distribution, tolerance, imaging, and antiviral and anticancer activity.

The aim of this study was to investigate the distribution, tolerance, and anticancer and antiviral activity of Zn-based physiometacomposites (PMCs). Manganese, iron, nickel and cobalt-doped ZnO, ZnS or ZnSe were synthesized. Cell uptake, distribution into 3D culture and mice, and biochemical and chemotherapeutic activity were studied by fluorescence/bioluminescence, confocal microscopy, flow cytometry, viability, antitumor and virus titer assays. Luminescence and inductively coupled plasma mass spectrometry analysis showed that nanoparticle distribution was liver >spleen >kidney >lung >brain, without tissue or blood pathology. Photophysical characterization as ex vivo tissue probes and LL37 peptide, antisense oligomer or aptamer delivery targeting RAS/Ras binding domain (RBD) was investigated. Treatment at 25 µg/ml for 48 h showed ≥98-99% cell viability, 3D organoid uptake, 3-log inhibition of ß-Galactosidase and porcine reproductive respiratory virus infection. Data support the preclinical development of PMCs for imaging and delivery targeting cancer and infectious disease.

Evaluation of Intraoperative Hand-Off Frequency, Duration, and Context: A Mixed Methods Analysis.

BACKGROUND: Hand-offs in the operating room contribute to poor communication, reduced team function, and may be poorly coordinated with other activities. Conversely, they may represent a missed opportunity for improved communication. We sought to better understand the coordination and impact of intraoperative hand-offs. METHODS: We prospectively audio-video (AV) recorded 10 operations and evaluated intraoperative hand-offs. Data collected included percentage of time team members were absent due to breaks, relationships between hand-offs and intraoperative events (incision, surgical counts), and occurrences of simultaneous hand-offs. We also identified announcement that a hand-off had occurred and anchoring, in which team members not involved in the hand-off participated and provided information. RESULTS: Spanning 2919 min of audio-video data, there were 74 hand-offs (range, 4-14 per case) totaling 225.2 min, representing 7.7% of time recorded. Thirty-two (45.1%) hand-offs were interrupted or delayed because of competing activities; eight hand-offs occurred during an instrument or laparotomy pad count. Six cases had simultaneous hand-offs; two cases had two episodes of simultaneous hand-offs. Eight hand-offs included an announcement. Seven included anchoring. Evaluating both temporary and permanent hand-offs, one or more original team members was absent for 40.7% of time recorded and >one team member was absent for 20.5% of time recorded. CONCLUSIONS: Intraoperative hand-offs are frequent and not well coordinated with intraoperative events including counts and other hand-offs. Anchoring and announced hand-offs occurred in a small proportion of cases. Future work must focus on optimizing timing, content, and participation in intraoperative hand-offs.

A BAC Transgene Expressing Human CFTR under Control of Its Regulatory Elements Rescues Cftr Knockout Mice.

Small-molecule modulators of cystic fibrosis transmembrane conductance regulator (CFTR) biology show promise in the treatment of cystic fibrosis (CF). A Cftr knockout (Cftr KO) mouse expressing mutants of human CFTR would advance in vivo testing of new modulators. A bacterial artificial chromosome (BAC) carrying the complete hCFTR gene including regulatory elements within 40.1 kb of DNA 5' and 25 kb of DNA 3' to the gene was used to generate founder mice expressing hCFTR. Whole genome sequencing indicated a single integration site on mouse chromosome 8 (8qB2) with ~6 gene copies. hCFTR+ offspring were bred to murine Cftr KO mice, producing hCFTR+/mCftr- (H+/m-) mice, which had normal survival, growth and goblet cell function as compared to wild-type (WT) mice. Expression studies showed hCFTR protein and transcripts in tissues typically expressing mCftr. Functionally, nasal potential difference and large intestinal short-circuit (Isc) responses to cAMP stimulation were similar in magnitude to WT mice, whereas small intestinal cAMP ΔIsc responses were reduced. A BAC transgenic mouse with functional hCFTR under control of its regulatory elements has been developed to enable the generation of mouse models of hCFTR mutations by gene editing for in vivo testing of new CF therapies.

Familiarity and Communication in the Operating Room.

BACKGROUND: Poor communication is implicated in many adverse events in the operating room (OR); however, many hospitals' scheduling practices permit unfamiliar operative teams. The relationship between unfamiliarity, team communication and effectiveness of communication is poorly understood. We sought to evaluate the relationship between familiarity, communication rates, and communication ineffectiveness of health care providers in the OR. MATERIALS AND METHODS: We performed purposive sampling of 10 open operations. For each case, six providers (anesthesiology attending, in-room anesthetist, circulator, scrub, surgery attending, and surgery resident) were queried about the number of mutually shared cases. We identified communication events and created dyad-specific communication rates. RESULTS: Analysis of 48 h of audio-video content identified 2570 communication events. Operations averaged 58.0 communication events per hour (range, 29.4-76.1). Familiarity was not associated with communication rate (P = 0.69) or communication ineffectiveness (P = 0.21). Cross-disciplinary dyads had lower communication rates than intradisciplinary dyads (P < 0.001). Anesthesiology-nursing, anesthesiology-surgery, and nursing-surgery dyad communication rates were 20.1%, 42.7%, and 57.3% the rate predicted from intradisciplinary dyads, respectively. In addition, cross-disciplinary dyad status was a significant predictor of having at least one ineffective communication event (P = 0.02). CONCLUSIONS: Team members do not compensate for unfamiliarity by increasing their verbal communication, and dyad familiarity is not protective against ineffective communication. Cross-disciplinary communication remains vulnerable in the OR suggesting poor crosstalk across disciplines in the operative setting. Further investigation is needed to explore these relationships and identify effective interventions, ensuring that all team members have the necessary information to optimize their performance.

Modeling Surgical Technical Skill Using Expert Assessment for Automated Computer Rating.

OBJECTIVE: Computer vision was used to predict expert performance ratings from surgeon hand motions for tying and suturing tasks. SUMMARY BACKGROUND DATA: Existing methods, including the objective structured assessment of technical skills (OSATS), have proven reliable, but do not readily discriminate at the task level. Computer vision may be used for evaluating distinct task performance throughout an operation. METHODS: Open surgeries was videoed and surgeon hands were tracked without using sensors or markers. An expert panel of 3 attending surgeons rated tying and suturing video clips on continuous scales from 0 to 10 along 3 task measures adapted from the broader OSATS: motion economy, fluidity of motion, and tissue handling. Empirical models were developed to predict the expert consensus ratings based on the hand kinematic data records. RESULTS: The predicted versus panel ratings for suturing had slopes from 0.73 to 1, and intercepts from 0.36 to 1.54 (Average R2 = 0.81). Predicted versus panel ratings for tying had slopes from 0.39 to 0.88, and intercepts from 0.79 to 4.36 (Average R2 = 0.57). The mean square error among predicted and expert ratings was consistently less than the mean squared difference among individual expert ratings and the eventual consensus ratings. CONCLUSIONS: The computer algorithm consistently predicted the panel ratings of individual tasks, and were more objective and reliable than individual assessment by surgical experts.

Cftr Modulates Wnt/ß-Catenin Signaling and Stem Cell Proliferation in Murine Intestine.

BACKGROUND & AIMS: Cystic fibrosis (CF) patients and CF mouse models have increased risk for gastrointestinal tumors. CF mice show augmented intestinal proliferation of unknown etiology and an altered intestinal environment. We examined the role of the cystic fibrosis transmembrane conductance regulator (Cftr) in Wnt/ß-catenin signaling, stem cell proliferation, and its functional expression in the active intestinal stem cell (ISC) population. Dysregulation of intracellular pH (pHi) in CF ISCs was investigated for facilitation of Wnt/ß-catenin signaling. METHODS: Crypt epithelia from wild-type (WT) and CF mice were compared ex vivo and in intestinal organoids (enteroids) for proliferation and Wnt/ß-catenin signaling by standard assays. Cftr in ISCs was assessed by immunoblot of sorted Sox9 enhanced green fluorescent protein(EGFP) intestinal epithelia and pHi regulation by confocal microfluorimetry of leucine-rich G-protein-coupled receptor 5 ISCs. Plasma membrane association of the Wnt transducer Dishevelled 2 (Dvl2) was assessed by fluorescence imaging of live enteroids from WT and CF mice crossed with Dvl2-EGFP/ACTB-tdTomato,-EGFP)Luo/J (RosamT/mG) mice. RESULTS: Relative to WT, CF intestinal crypts showed an ∼30% increase in epithelial and Lgr5+ ISC proliferation and increased Wnt/ß-catenin signaling. Cftr was expressed in Sox9EGFPLo ISCs and loss of Cftr induced an alkaline pHi in ISCs. CF crypt-base columnar cells showed a generalized increase in plasma membrane Dvl2-EGFP association as compared with WT. Dvl2-EGFP membrane association was charge- and pH-dependent and increased in WT crypt-base columnar cells by Cftr inhibition. CONCLUSIONS: CF intestine shows increased ISC proliferation and Wnt/ß-catenin signaling. Loss of Cftr increases pHi in ISCs, which stabilizes the plasma membrane association of the Wnt transducer Dvl, likely facilitating Wnt/ß-catenin signaling. Absence of Cftr-dependent suppression of ISC proliferation in the CF intestine may contribute to increased risk for intestinal tumors.

A Statewide Surgical Coaching Program Provides Opportunity for Continuous Professional Development.

OBJECTIVE: We sought to develop and evaluate a video-based coaching program for board-eligible/certified surgeons. SUMMARY BACKGROUND DATA: Multiple disciplines utilize coaching for continuous professional development; however, coaching is not routinely employed for practicing surgeons. METHODS: Peer-nominated surgeons were trained as coaches then paired with participant surgeons. After setting goals, each coaching pair reviewed video-recorded operations performed by the participating surgeon. Coaching sessions were audio-recorded, transcribed, and coded to identify topics discussed. The effectiveness with which our coaches were able to utilize the core principles and activities of coaching was evaluated using 3 different approaches: self-evaluation; evaluation by the participants; and assessment by the study team. Surveys of participating surgeons and coach-targeted interviews provided general feedback on the program. All measures utilized a 5-point Likert scale format ranging from 1 (low) to 5 (high). RESULTS: Coach-participant surgeon pairs targeted technical, cognitive, and interpersonal aspects of performance. Other topics included managing intraoperative stress. Mean objective ratings of coach effectiveness was 3.1 ±â€Š0.7, ranging from 2.0 to 5.0 on specific activities of coaching. Subjective ratings by coaches and participants were consistently higher. Coaches reported that the training provided effectively prepared them to facilitate coaching sessions. Participants were similarly positive about interactions with their coaches. Identified barriers were related to audio-video technology and scheduling of sessions. Overall, participants were satisfied with their experience (mean 4.4 ±â€Š0.7) and found the coaching program valuable (mean 4.7 ±â€Š0.7). CONCLUSIONS: This is the first report of cross-institutional surgical coaching for the continuous professional development of practicing surgeons, demonstrating perceived value among participants, as well as logistical challenges for implementing this evidence-based program. Future research is necessary to evaluate the impact of coaching on practice change and patient outcomes.