Preclinical evidence for employing MEK inhibition in NRAS mutated pediatric gastroenteropancreatic neuroendocrine-like tumors.

BACKGROUND: Pediatric gastroenteropancreatic neuroendocrine tumors are exceedingly rare, resulting in most pediatric treatment recommendations being based on data derived from adults. Trametinib is a kinase inhibitor that targets MEK1/2 and has been employed in the treatment of cancers harboring mutations in the Ras pathway. METHODS: We utilized an established human pediatric gastroenteropancreatic neuroendocrine-like tumor patient-derived xenograft (PDX) with a known NRAS mutation to study the effects of MEK inhibition. We evaluated the effects of trametinib on proliferation, motility, and tumor growth in vivo. We created an intraperitoneal metastatic model of this PDX, characterized both the phenotype and the genotype of the metastatic PDX and again, investigated the effects of MEK inhibition. RESULTS: We found target engagement with decreased ERK1/2 phosphorylation with trametinib treatment. Trametinib led to decreased in vitro cell growth and motility, and decreased tumor growth and increased animal survival in a murine flank tumor model. Finally, we demonstrated that trametinib was able to significantly decrease gastroenteropancreatic neuroendocrine intraperitoneal tumor metastasis. CONCLUSIONS: The results of these studies support the further investigation of MEK inhibition in pediatric NRAS mutated solid tumors.

Diabetes Increases Median Nerve Cross-Sectional Area but Not Disease Severity in Patients with Carpal Tunnel Syndrome.

Background: Ultrasonography (US) is a useful diagnostic modality for diagnosis of carpal tunnel syndrome (CTS). Diabetes mellitus is increasingly prevalent and is a risk factor for CTS. Given the increasing use of US in the diagnosis of CTS, our goal was to evaluate the influence of diabetes on CTS severity and the cross-sectional area (CSA) of the median nerve in patients with CTS. Methods: Patients with clinically diagnosed CTS were seen in the outpatient setting from October 2014 to February 2021. Median nerve CSA and patient reported severity measures were obtained: Boston Carpal Tunnel Syndrome Questionnaire (BCTSQ) and CTS-6. For patients with diabetes, additional parameters were collected including most recent A1c, insulin pharmacotherapy, and polypharmacy. Results: Ninety-nine patients (122 nerves) without diabetes and 55 patients (82 nerves) with diabetes were recruited for the study. Patients in the diabetes group were more obese and older and had a significantly increased median nerve CSA compared with patients without diabetes. Obesity was associated with higher median nerve CSA in all patients but not in patients with diabetes. There was no difference in disease severity in patients with and without diabetes as reported by BCTSQ or CTS-6 scores. In patients with diabetes, there was significantly decreased median nerve CSA with A1c of 6.5 or higher and a trend to decreased CSA with polypharmacy. There was no influence of insulin therapy on median nerve CSA. Conclusion: Diabetes is associated with higher median nerve CSA in patients with CTS of similar disease severity. The increased median nerve CSA in patients with diabetes may be reflective of diabetes-related microvascular changes. Interestingly, the trend to decreased median nerve CSA in patients with suboptimal diabetic control (A1c ≥ 6.5) may suggest eventual degenerative changes to the median nerve. In summary, clinicians should be cautious with interpreting a larger median nerve CSA as more severe CTS in patients with diabetes. Level of Evidence: Level 3 Diagnostic.

Evaluation of Timing of Antimicrobial Surgical Prophylaxis on Rates of Surgical Site Infections.

Background: Surgical site infections (SSIs) are common healthcare-associated infections, and national guidelines recommend that antimicrobial prophylaxis (AP) be administered 60 min prior to incision. However, there are limited data regarding the "most optimal" time for administration within the 60-min window. Patients and Methods: This was a multicenter, retrospective study of adult (≥18-year-old) patients that underwent an abdominal hysterectomy, colorectal surgery, or craniotomy and received AP within 60 min of incision. Incidence of SSI was compared between patients who received AP 0-30 versus 31-60 min of incision. In addition, a predefined subgroup analysis evaluated incidence of SSI for 15-min intervals within the 60-min timeframe. Results: Of the 277 patients included in the primary analysis, 233 (84.1%) and 44 (15.9%) received AP 0-30 min and 31-60 min prior to incision, respectively. SSIs were documented in 6.0% (14/233) versus 4.5% (2/44) of patients in the primary analysis (p = 0.703). In the secondary analysis, 137 (49.5%), 95 (34.3%), 34 (12.3%), and 11 (4.0%) patients received AP 0-15, 16-30, 31-45, and 46-60 min prior to incision, respectively. There was no difference in incidence of SSIs among the 15-min intervals (4.4% vs. 8.4% vs. 2.9% vs. 9.1%, p = 0.487). Of the 16 patients in this study that incurred a SSI, 5 patients had positive cultures, of which 3 contained bacteria that proved to be resistant to the antibiotic used for AP. Conclusions: The results of our analysis support current national guidelines. Future investigation of different intervals (e.g., AP 15-45 min prior to incision) may be beneficial on the basis of pharmacokinetics of routinely prescribed AP.

Does Time to Asystole in Donors After Circulatory Death Impact Recipient Outcome in Liver Transplantation?

BACKGROUND: The agonal phase can vary following treatment withdrawal in donor after circulatory death (DCD). There is little evidence to support when procurement teams should stand down in relation to donor time to death (TTD). We assessed what impact TTD had on outcomes following DCD liver transplantation. METHODS: Data were extracted from the UK Transplant Registry on DCD liver transplant recipients from 2006 to 2021. TTD was the time from withdrawal of life-sustaining treatment to asystole, and functional warm ischemia time was the time from donor systolic blood pressure and/or oxygen saturation falling below 50 mm Hg and 70%, respectively, to aortic perfusion. The primary endpoint was 1-y graft survival. Potential predictors were fitted into Cox proportional hazards models. Adjusted restricted cubic spline models were generated to further delineate the relationship between TTD and outcome. RESULTS: One thousand five hundred fifty-eight recipients of a DCD liver graft were included. Median TTD in the entire cohort was 13 min (interquartile range, 9-17 min). Restricted cubic splines revealed that the risk of graft loss was significantly greater when TTD ≤14 min. After 14 min, there was no impact on graft loss. Prolonged hepatectomy time was significantly associated with graft loss (hazard ratio, 1.87; 95% confidence interval, 1.23-2.83; P = 0.003); however, functional warm ischemia time had no impact (hazard ratio, 1.00; 95% confidence interval, 0.44-2.27; P > 0.9). CONCLUSIONS: A very short TTD was associated with increased risk of graft loss, possibly because of such donors being more unstable and/or experiencing brain stem death as well as circulatory death. Expanding the stand down times may increase the utilization of donor livers without significantly impairing graft outcome.

The Australian health workforce: Disproportionate shortfalls in small rural towns.

INTRODUCTION: The distribution of health care workers differs greatly across Australia, which is likely to impact health delivery. OBJECTIVE: To examine demographic and workplace setting factors of doctors, nurses and midwives, and allied health professionals across Modified Monash Model (MMM) regions and identify factors associated with shortfalls in the health care workforce. DESIGN: Descriptive cross-sectional analysis. The study included all health professionals who were registered with the Australian Health Practitioner Regulation Agency in 2021, and who were working in Australia in their registered profession. The study examined number of registrations and full-timed equivalent (FTE) registrations per MMM region classification, adjusted for population. Associated variables included age, gender, origin of qualification, Indigenous status and participation in the private or public (including government, non-government organisation and not-for-profit organisations) sectors. FINDINGS: Data were available for 31 221 general practitioners, 77 277 other doctors, 366 696 nurses and midwives, and 195 218 allied health professionals. The lowest FTE per 1000 people was seen in MM5 regions for general practitioners, other doctors, nurses and midwives, and allied health professionals. Demographic factors were mostly consistent across MM regions, although MM5 regions had a higher percentage of nurses and midwives and allied health professionals aged 55 and over. In the private sector, FTE per 1000 people was lowest in MM5-7 regions. In the public sector, FTE per 1000 people was lowest in MM5 regions. DISCUSSION: A disproportionate shortfall of health workers was seen in MM5 regions. This shortfall appears to be primarily due to low FTE per capita of private sector workers compared with MM1-4 regions and a low FTE per capita of public sector workers compared with MM6-7 regions. CONCLUSION: In Australia, small rural towns have the lowest number of health care workers per capita which is likely to lead to poor health outcomes for those regions.

PIM Kinase Inhibition Sensitizes Neuroblastoma to Doxorubicin.

BACKGROUND: Chemoresistance contributes to relapse in high-risk neuroblastoma. Cancer cells acquire resistance through multiple mechanisms, including drug efflux pumps. In neuroblastoma, multidrug resistance-associated protein-1 (MRP1/ABCC1) efflux pump expression correlates with worse outcomes. These pumps are regulated by PIM kinases, a family of serine-threonine kinases, overexpressed in neuroblastoma. We hypothesized PIM kinase inhibition would sensitize neuroblastoma cells by modulating MRP1. METHODS: Kocak database query evaluated ABCC1, PIM1, PIM2, and PIM3 expression in neuroblastoma patients. SK-N-AS and SK-N-BE(2) cells were treated with doxorubicin or the pan-PIM kinase inhibitor, AZD1208. Flow cytometry assessed intracellular doxorubicin accumulation. AlamarBlue assay measured viability. The lethal dose 50% (LD50) of each drug and combination indices (CI) were calculated and isobolograms constructed to determine synergy. RESULTS: Kocak database query demonstrated positive correlation between PIM genes and ABCC1. PIM kinase inhibition increased intracellular doxorubicin accumulation in both cell lines, suggesting PIM kinase regulation of MRP1. Isobolograms showed synergy between AZD1208 and doxorubicin. CONCLUSIONS: The correlation between PIM and ABCC1 gene expression suggests PIM kinases may contribute to neuroblastoma chemotherapeutic resistance. PIM kinase inhibition increased intracellular doxorubicin accumulation. Combination treatment with AZD1208 and doxorubicin decreased neuroblastoma cell viability in a synergistic fashion. These findings support further investigations of PIM kinase inhibition in neuroblastoma. TYPE OF STUDY: Basic Science Research. LEVEL OF EVIDENCE: NA.

DNA binding analysis of rare variants in homeodomains reveals homeodomain specificity-determining residues.

Homeodomains (HDs) are the second largest class of DNA binding domains (DBDs) among eukaryotic sequence-specific transcription factors (TFs) and are the TF structural class with the largest number of disease-associated mutations in the Human Gene Mutation Database (HGMD). Despite numerous structural studies and large-scale analyses of HD DNA binding specificity, HD-DNA recognition is still not fully understood. Here, we analyze 92 human HD mutants, including disease-associated variants and variants of uncertain significance (VUS), for their effects on DNA binding activity. Many of the variants alter DNA binding affinity and/or specificity. Detailed biochemical analysis and structural modeling identifies 14 previously unknown specificity-determining positions, 5 of which do not contact DNA. The same missense substitution at analogous positions within different HDs often exhibits different effects on DNA binding activity. Variant effect prediction tools perform moderately well in distinguishing variants with altered DNA binding affinity, but poorly in identifying those with altered binding specificity. Our results highlight the need for biochemical assays of TF coding variants and prioritize dozens of variants for further investigations into their pathogenicity and the development of clinical diagnostics and precision therapies.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here, we identify the domains of LRMP essential for this regulation, show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we identified the initial 227 residues of LRMP and the N-terminus of HCN4 as necessary for LRMP to associate with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Finally, we demonstrated that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of five residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating, most likely via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Implementing Tourniquet Conversion Guidelines for Civilian EMS and Prehospital Organizations : A Case Report and Review.

Since the first documented use of a tourniquet in 1674, the popularity of tourniquets has waxed and waned. During recent wars and more recently in Emergency Medical Services systems, the tourniquet has been proven to be a valuable tool in the treatment of life-threatening hemorrhage. However, tourniquet use is not without risk, and several studies have demonstrated adverse events and morbidity associated with tourniquet use in the prehospital setting, particularly when left in place for more than 2 h. Consequently, the US military's Committee on Tactical Combat Casualty Care has recommended guidelines for prehospital tourniquet conversion to reduce the risk of adverse events associated with tourniquets once the initial hemorrhage has been controlled. Emergency Medical Services systems that operate in rural, frontier, and austere environments, especially those with transport times to definitive care that routinely exceed 2 h, may consider implementing similar tourniquet conversion guidelines.

Elbow Uncemented Hemiarthroplasty: Surgical Technique.

Management of elbow arthritis in younger and higher demand patients is challenging and may benefit from a distal humerus hemiarthroplasty that employs a noncemented method of implant fixation and stabilizes the elbow through ligament reconstruction. By not replacing both articulating surfaces, hardware longevity may be improved. We describe a novel system that may be indicated for the treatment of posttraumatic or primary osteoarthritis of the distal humerus. The step-by-step technique for surgical implantation of this uncemented distal humerus hemiarthroplasty is described and illustrated.

The impact of time to death in donors after circulatory death on recipient outcome in simultaneous pancreas-kidney transplantation.

The time to arrest donors after circulatory death is unpredictable and can vary. This leads to variable periods of warm ischemic damage prior to pancreas transplantation. There is little evidence supporting procurement team stand-down times based on donor time to death (TTD). We examined what impact TTD had on pancreas graft outcomes following donors after circulatory death (DCD) simultaneous pancreas-kidney transplantation. Data were extracted from the UK transplant registry from 2014 to 2022. Predictors of graft loss were evaluated using a Cox proportional hazards model. Adjusted restricted cubic spline models were generated to further delineate the relationship between TTD and outcome. Three-hundred-and-seventy-five DCD simultaneous kidney-pancreas transplant recipients were included. Increasing TTD was not associated with graft survival (adjusted hazard ratio HR 0.98, 95% confidence interval 0.68-1.41, P = .901). Increasing asystolic time worsened graft survival (adjusted hazard ratio 2.51, 95% confidence interval 1.16-5.43, P = .020). Restricted cubic spline modeling revealed a nonlinear relationship between asystolic time and graft survival and no relationship between TTD and graft survival. We found no evidence that TTD impacts pancreas graft survival after DCD simultaneous pancreas-kidney transplantation; however, increasing asystolic time was a significant predictor of graft loss. Procurement teams should attempt to minimize asystolic time to optimize pancreas graft survival rather than focus on the duration of TTD.

A comparison of rural and regional work locations and speciality choices between graduates from the University of Wollongong and all Australian medical schools using the Medical Schools Outcomes Database.

INTRODUCTION: The shortfall in medical workers in rural and remote Australia has led to health discrepancies in these regions. The University of Wollongong's medical program was designed to encourage graduates to work in these regions to address this shortfall. OBJECTIVE: To compare rural and regional locations of work and choices of speciality between University of Wollongong's graduates and graduates from all Australian universities. DESIGN: We conducted a longitudinal analysis on data available from the Medical Schools Outcome database, with graduate exit surveys linked to registrations of location and speciality. Rural and remote locations were identified as MM2-7 regions using the Modified Monash Model. In total, 716 graduates from the University of Wollongong and 26 915 graduates from all Australian medical schools completed the MSOD exit survey in 2010-2021 and registered with the Australian Health Practitioner Regulation Agency in 2022. The main outcome was the relative likelihood (relative risk) of cohorts working in rural and regional areas and of cohorts choosing general practice as their speciality. FINDINGS: University of Wollongong's medical graduates were 1.51 times or 51% more likely to work in regional or rural areas (RR 1.51, 95% CI 1.34 to 1.71, p < 0.0001). Respondents who were 10 or more years post graduation were 1.57 times or 57% more likely to specialise in general practice than all other Australian medical graduates (RR 1.57 95% CI: 1.40 to 1.79, p < 0.0001). DISCUSSION: The University of Wollongong's medical school is producing graduates to meet Australia's rural health workforce needs. This may be due to a higher intake of rural students, and a higher percentage of students taking rural placements. CONCLUSIONS: Rural health workforce needs can be addressed through rural-focussed education strategies.

CDK4/6 Inhibition With Lerociclib is a Potential Therapeutic Strategy for the Treatment of Pediatric Sarcomas.

BACKGROUND: Sarcomas are a heterogenous collection of bone and soft tissue tumors. The heterogeneity of these tumors makes it difficult to standardize treatment. CDK 4/6 inhibitors are a family of targeted agents which limit cell cycle progression and have been shown to be upregulated in sarcomas. In the current preclinical study, we evaluated the effects of lerociclib, a CDK4/6 inhibitor, on pediatric sarcomas in vitro and in 3D bioprinted tumors. METHODS: The effects of lerociclib on viability, proliferation, cell cycle, motility, and stemness were assessed in established sarcoma cell lines, U-2 OS and MG-63, as well as sarcoma patient-derived xenografts (PDXs). 3D printed biotumors of each of the U-2 OS, MG-63, and COA79 cells were utilized to study the effects of lerociclib on tumor growth ex vivo. RESULTS: CDK 4/6, as well as the intermediaries retinoblastoma protein (Rb) and phosphorylated Rb were identified as targets in the four sarcoma cell lines. Lerociclib treatment induced cell cycle arrest, decreased proliferation, motility, and stemness of sarcoma cells. Treatment with lerociclib decreased sarcoma cell viability in both traditional 2D culture as well as 3D bioprinted microtumors. CONCLUSIONS: Inhibition of CDK 4/6 activity with lerociclib was efficacious in traditional 2D sarcoma cell culture as well as in 3D bioprints. Lerociclib holds promise and warrants further investigation as a novel therapeutic strategy for management of these heterogenous groups of tumors.

Subjective and Objective Differences in Patients with Unilateral and Bilateral Carpal Tunnel Syndrome and the Role of Obesity in Syndrome Severity.

BACKGROUND: It remains unclear whether physiologic differences exist in musculoskeletal ultrasound nerve measurements when comparing bilateral and unilateral carpal tunnel syndrome (CTS) patients. Similarly, the influence of body mass index on CTS severity is not well characterized. METHODS: Unilateral and bilateral CTS patients were seen from October of 2014 to February of 2021. Obese and nonobese CTS patients were compared. Median nerve cross-sectional area (CSA), Boston Carpal Tunnel Syndrome Questionnaire (BCTSQ), and six-item Carpal Tunnel Symptom Score (CTS-6) measures were obtained. Nerve conduction studies recorded distal motor latency (DML) and distal sensory latency (DSL). Statistical analysis used Wilcoxon signed rank testing for paired continuous variables, Mann-Whitney U testing for nonpaired continuous variables, and chi-square testing for continuous variables, with a significance level of P < 0.05. RESULTS: A total of 109 (218 nerves) bilateral and 112 (112 nerves) unilateral CTS patients were reviewed. Bilateral patients had larger median nerve CSAs on their more symptomatic side, when defined by BCTSQ score ( P < 0.0001), CTS-6 score ( P < 0.0001), DML ( P < 0.0001), and DSL ( P < 0.01). Bilateral patients also had higher symptom severity scale ( P < 0.01) and DSL ( P < 0.001) outcomes compared with unilateral patients. Obese patients had higher median nerve CSA ( P < 0.01), prolonged DML, and prolonged DSL ( P < 0.0001) values despite similar CTS severity (BCTSQ and CTS-6). CONCLUSIONS: Ultrasound identifies the more symptomatic side in bilateral patients, which correlates with increasing severity (NCS and BCTSQ). Obesity increases median nerve CSA and prolongs nerve conduction studies without influencing CTS severity. This information can be used when considering which diagnostic test to order for CTS.

LRMP inhibits cAMP potentiation of HCN4 channels by disrupting intramolecular signal transduction.

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation. We show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating. And we demonstrate that multiple unique regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we showed that the initial 227 residues of LRMP and the N-terminus of HCN4 are necessary for LRMP to interact with HCN4. We found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. And we demonstrate that LRMP-regulation can be conferred to HCN2 by addition of the HCN4 N-terminus along with mutation of 5 residues in the S5 region and C-linker to the cognate HCN4 residues. Taken together, these results suggest that LRMP inhibits HCN4 through an isoform-specific interaction involving the N-terminals of both proteins that prevents the transduction of cAMP binding into a change in channel gating via an HCN4-specific orientation of the N-terminus, C-linker, and S4-S5 linker.

Static stability of novel uncemented elbow hemiarthroplasty stabilized with ligament reconstruction.

BACKGROUND: The purpose of this study was to create a model to simulate treatment of unreconstructable distal humerus fractures with hemiarthroplasty. Stability was restored with a latest plate-system that simultaneously tensions medial and lateral collateral ligament grafts. MATERIALS AND METHODS: Static varus and valgus elbow stability was tested in 11 cadaver elbows with intact ligaments and capsule at 5 flexion angles (0°, 30°, 60°, 90°, 120°). The elbows were then destabilized via release of all ligaments and capsular attachments. The distal humerus articular cartilage was excised and replaced with an uncemented hemiarthroplasty. Ligament reconstruction was subsequently performed, and elbow stability was measured and compared to the native state. Dimensions of the hemiarthroplasty component were compared to native elbow dimensions to assess and quantify any existing relationship to elbow stability. RESULTS: A hemiarthroplasty was implanted in all specimens. A size mismatch occurred between the distal humerus trochlea and the olecranon fossa in all specimens and averaged 6.3 mm. Following ligament reconstruction, specimens reproduced the flexion angle-dependent stability of native elbows to both varus and valgus stress. On the medial side, elbow joint stability in mid-flexion was approximately 7% tighter after hemiarthroplasty. Laterally, the elbow was approximately 15% tighter after hemiarthroplasty and demonstrated peak stability in full flexion. The 3 assessed hemiarthroplasty components and bony dimensions did not exhibit any relationship between implant-bone mismatch and elbow stability after ligamentous reconstruction. CONCLUSION: Cadaveric elbow specimens underwent uncemented hemiarthroplasty with soft tissue stabilization with a novel technique for ligament reconstruction. Following hemiarthroplasty and ligament reconstruction, these specimens maintained secure fixation between ligament and bone. Static stability was maintained at varying degrees of elbow flexion regardless of variable mismatch between the hemiarthroplasty component and the native olecranon fossa.