Albumin promoter-driven FlpO expression induces efficient genetic recombination in mouse liver.

Tissue-specific gene manipulations are widely used in genetically engineered mouse models. A single recombinase system, such as the one using Alb-Cre, has been commonly used for liver-specific genetic manipulations. However, most diseases are complex, involving multiple genetic changes and various cell types. A dual recombinase system is required for conditionally modifying different genes sequentially in the same cell or inducing genetic changes in different cell types within the same organism. A FlpO cDNA was inserted between the last exon and 3'-UTR of the mouse albumin gene in a bacterial artificial chromosome (BAC-Alb-FlpO). The founders were crossed with various reporter mice to examine the efficiency of recombination. Liver cancer tumorigenesis was investigated by crossing the FlpO mice with FSF-KrasG12D mice and p53frt mice (KPF mice). BAC-Alb-FlpO mice exhibited highly efficient recombination capability in both hepatocytes and intrahepatic cholangiocytes. No recombination was observed in the duodenum and pancreatic cells. BAC-Alb-FlpO-mediated liver-specific expression of mutant KrasG12D and conditional deletion of p53 gene caused the development of liver cancer. Remarkably, liver cancer in these KPF mice manifested a distinctive mixed hepatocellular carcinoma and cholangiocarcinoma phenotype. A highly efficient and liver-specific BAC-Alb-FlpO mouse model was developed. In combination with other Cre lines, different genes can be manipulated sequentially in the same cell, or distinct genetic changes can be induced in different cell types of the same organism.NEW & NOTEWORTHY A liver-specific Alb-FlpO mouse line was generated. By coupling it with other existing CreERT or Cre lines, the dual recombinase approach can enable sequential gene modifications within the same cell or across various cell types in an organism for liver research through temporal and spatial gene manipulations.

PTEN Deficiency Induces an Extrahepatic Cholangitis-Cholangiocarcinoma Continuum via Aurora kinase A in Mice.

BACKGROUND & AIMS: The PTEN-AKT pathway is frequently altered in extrahepatic cholangiocarcinoma (eCCA). We aim to evaluate the role of PTEN in the pathogenesis of eCCA and find novel therapies for this disease. METHODS: The Pten gene in the biliary epithelial cells were genetically deleted using the Cre-loxp system. The pathologies were evaluated both macroscopically and histologically. The characteristics were further analyzed by immunohistochemistry (IHC), RT-PCR, cell culture, and RNAseq. Some features were compared to those in human eCCA samples. Further mechanistic studies utilized the conditional knockout of Trp53 and Aurora kinase A (Aurka) genes. Experimental therapy was tested using an Aurka inhibitor. RESULTS: We observed that genetic deletion of the Pten gene in the extrahepatic biliary epithelium and peri-ductal glands initiated sclerosing cholangitis-like lesions in mice, resulting in enlarged and distorted extrahepatic bile ducts in mice as early as one month old. Histologically, these lesions exhibited increased epithelial proliferation, inflammatory cell infiltration, and fibrosis. With aging, the lesions progressed from low-grade dysplasia to invasive carcinoma. Trp53 inactivation further accelerated the disease progression, potentially through downregulating senescence. Further mechanistic studies showed that both human and mouse eCCA showed high expressions of AURKA. Notably, the genetic deletion of Aurka completely eliminated Pten deficiency-induced extrahepatic bile duct lesions. Furthermore, pharmacological inhibition of Aurka alleviated disease progression. CONCLUSIONS: Pten deficiency in extrahepatic cholangiocytes and peribiliary glands led to a cholangitis-to-cholangiocarcinoma continuum through an Aurka-dependent manner. These findings offer new insights into preventive and therapeutic interventions for extrahepatic CCA. IMPACT AND IMPLICATIONS: The aberrant PTEN-PI3K-AKT signaling pathway is commonly observed in human extrahepatic cholangiocarcinoma (eCCA), a disease with a poor prognosis. In our study, we developed a mouse model mimicking cholangitis to eCCA progression by conditionally deleting the Pten gene via Pdx1-Cre in epithelial cells and peribiliary glands of the extrahepatic biliary duct. The conditional Pten deletion in these cells led to cholangitis, which gradually advanced to dysplasia, ultimately resulting in eCCA. The loss of Pten heightened Akt signaling, cell proliferation, inflammation, fibrosis, DNA damage, epigenetic signaling, epithelial-mesenchymal transition (EMT), cell dysplasia, and cellular senescence. Genetic deletion or pharmacological inhibition of Aurka successfully halted the disease progression. This model shall be valuable for testing novel therapies and unraveling the mechanisms of eCCA tumorigenesis.

Age-related changes in hematological and biochemical profiles of Wistar rats.

BACKGROUND: Wistar rats are extensively used as the model for assessing toxicity and efficacy in preclinical research. Hematological and biochemical laboratory data are essential for evaluating specific variations in the physiological and functional profile of a laboratory animal. Establishing hematological and biochemical reference values for Wistar (han) rats at various age intervals was the goal of this work. Male and female Wistar rats (n = 660) of ages 6-8 weeks, 10-14 weeks and > 6 months were used in the experiment. Blood and serum were collected from these rats under fasting conditions. RESULTS: We observed that the majority of hematological and biochemical parameters were significantly influenced by sex and age. Hematological changes were significantly correlated to aging were increased red blood cells, hemoglobin, hematocrit, neutrophils, monocytes and eosinophils in both sexes, as well as decreased platelet, mean corpuscular volume, mean corpuscular hemoglobin and lymphocytes in both sexes. White blood cells of male rats were considerably higher than those of female rats in all age ranges. For biochemistry, increase in glucose, total protein and creatinine were seen in both sexes, along with increases in urea in females and alanine aminotransferase in males. Age was significantly associated with decreased alkaline phosphatase in both sexes. CONCLUSIONS: When using Wistar rats as a model, these reference values may be useful in evaluating the results.

Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

BACKGROUND: Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. METHODS: Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. RESULTS: Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. CONCLUSION: A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

Higher 2-Year Cumulative Incidence of Mental Health Disorders Following Irrigation and Debridement in Primary Lumbar Fusion.

Introduction: Spinal fusion is an operation that is employed to treat spinal diseases. Surgical site infection (SSI) after lumbar fusion (LF) is a postoperative complication. SSI is treated with irrigation and debridement (I&D), which requires readmittance following discharge or prolonged hospital stays, which are deleterious to patients' mental health. The long-term relationship between treating SSI with I&D and patients' mental health is still understudied. Methods: Using the Mariner dataset from the PearlDiver Patient Records Database using Current Procedural Terminology and International Classification of Diseases procedure codes, retrospective cohort analysis was carried out. This study involved 445,480 patients who underwent LF with at least 2-year follow-up and were followed up for 2 years. Of the patients, 2,762 underwent I&D. Using univariate analysis employing Pearson Chi-square and Student t-test, where appropriate (Table 1), patient demographics between cohorts were gathered. 2-year cumulative incidence (CI) between LF and I&D cohorts was calculated using Kaplan-Meier analysis (Fig. 1, 2, 3). Cox proportional hazards were employed to observe significant differences in CI rates (Table 2). Results: For patients who received I&D, 2-year CI depression (HR: 1.72; 95% CI: 1.49-1.99; P<0.001) and stress (HR: 1.35; 95% CI: 1.02-1.79; P=0.035) rates were significantly higher than for those who did not. There was no statistically significant difference in 2-year CI anxiety rates between cohorts (HR: 0.92; 95% CI: 0.58-1.46; P=0.719). Conclusions: In conclusion, 16.8% of patients developed new-onset depression 2 years following I&D, in comparison to 10.3% of those who underwent LF. Patients who underwent I&D following LF were significantly more likely to experience depression and stress. To mitigate negative mental health outcomes, mental health services should be available to patients who underwent surgery.

Structure and Funding of Clinical Informatics Fellowships: A National Survey of Program Directors.

BACKGROUND: In 2011, the American Board of Medical Specialties established clinical informatics (CI) as a subspecialty in medicine, jointly administered by the American Board of Pathology and the American Board of Preventive Medicine. Subsequently, many institutions created CI fellowship training programs to meet the growing need for informaticists. Although many programs share similar features, there is considerable variation in program funding and administrative structures. OBJECTIVES: The aim of our study was to characterize CI fellowship program features, including governance structures, funding sources, and expenses. METHODS: We created a cross-sectional online REDCap survey with 44 items requesting information on program administration, fellows, administrative support, funding sources, and expenses. We surveyed program directors of programs accredited by the Accreditation Council for Graduate Medical Education between 2014 and 2021. RESULTS: We invited 54 program directors, of which 41 (76%) completed the survey. The average administrative support received was $27,732/year. Most programs (85.4%) were accredited to have two or more fellows per year. Programs were administratively housed under six departments: Internal Medicine (17; 41.5%), Pediatrics (7; 17.1%), Pathology (6; 14.6%), Family Medicine (6; 14.6%), Emergency Medicine (4; 9.8%), and Anesthesiology (1; 2.4%). Funding sources for CI fellowship program directors included: hospital or health systems (28.3%), clinical departments (28.3%), graduate medical education office (13.2%), biomedical informatics department (9.4%), hospital information technology (9.4%), research and grants (7.5%), and other sources (3.8%) that included philanthropy and external entities. CONCLUSION: CI fellowships have been established in leading academic and community health care systems across the country. Due to their unique training requirements, these programs require significant resources for education, administration, and recruitment. There continues to be considerable heterogeneity in funding models between programs. Our survey findings reinforce the need for reformed federal funding models for informatics practice and training.

Predictors of Survival in Patients With Hepatocellular Cancer Receiving Atezolizumab and Bevacizumab.

OBJECTIVES: In randomized clinical trials in patients with hepatocellular cancer (HCC), combination therapy with atezolizumab and bevacizumab (Atezo-Bev) prolonged survival, and these treatments have become the standard first-line therapy for advanced HCC. However, clinical trials may not reflect real-life clinical practice due to treatment selection criteria. Thus, our aim was to understand predictors of HCC outcomes with these treatments in a real-world, multicenter setting. METHODS: A retrospective review of all patients 18 years of age or older treated for advanced primary liver cancer between February 2020 and August 2022 was conducted to assess the relationship between overall survival and clinical and biochemical variables before or during treatment. Univariate and multivariate Cox regression survival analyses were performed to identify predictors of survival following treatment. RESULTS: One hundred and eleven eligible patients with unresectable HCC received Atezo-Bev over a consecutive 30-month period. Cox regression identified several significant ( P <0.05) predictors of survival, including pretreatment albumin (hazard ratios [HR]: 0.2; CI: 0.1-0.4), total bilirubin (HR: 1.3; CI: 1.2-1.5), and international normalized ratio (HR: 5.6; CI: 2.5-12.5). In multivariate analyses, these were significantly associated as predictors of mortality, and patients with pretreatment albumin <3.5 mg/dL had significantly lower survival than those ≥3.5 (153 vs. 522 d, P <0.0001). CONCLUSIONS: Pretreatment hypoalbuminemia, high bilirubin, and biochemical tests indicative of hepatic or renal dysfunction can independently predict short-term mortality in advanced HCC patients receiving Atezo-Bev.

Geospatial analysis of cyanobacterial exposure and liver cancer in the contiguous United States.

BACKGROUND AND AIMS: Cyanobacteria are commonly found in water bodies and their production of hepatotoxins can contribute to liver damage. However, the population health effects of cyanobacteria exposure (CE) are unknown. Our objectives were to determine the effect of chronic exposure to cyanobacteria through proximity to water bodies with high cyanobacteria counts on the incidence and mortality of liver cancers, as well as to identify location-based risk factors. APPROACH AND RESULTS: Across the contiguous United States, regions with high cyanobacteria counts in water bodies were identified using satellite remote sensing data. The data were geospatially mapped to county boundaries, and disease mortality and incidence rates were analyzed. Distinctive spatial clusters of CE and mortality related to liver diseases or cancer were identified. There was a highly significant spatial association between CE, liver disease, and liver cancer but not between CE and all cancers. Hot spots of CE and mortality were identified along the Gulf of Mexico, eastern Texas, Louisiana, and Florida, and cold spots across the Appalachians. The social vulnerability index was identified as a major location-based determinant by logistic regression, with counties in the fourth or fifth quintiles having the highest prevalence of hot spots of CE and mortality from liver cancer. CONCLUSIONS: These findings emphasize the importance of environmental exposure to cyanobacteria as a location-based determinant of mortality from liver cancer. Public health initiatives addressing CE may be considered to reduce mortality, particularly in areas of high social vulnerability.

Engineering Cell-Derived Nanovesicles for Targeted Immunomodulation.

Extracellular vesicles (EVs) show promise for targeted drug delivery but face production challenges with low yields. Cell-derived nanovesicles (CDNVs) made by reconstituting cell membranes could serve as EV substitutes. In this study, CDNVs were generated from mesenchymal stem cells by extrusion. Their proteomic composition, in vitro and in vivo toxicity, and capacity for loading RNA or proteins were assessed. Compared with EVs, CDNVs were produced at higher yields, were comprised of a broader range of proteins, and showed no detrimental effects on cell proliferation, DNA damage, or nitric oxide production in vitro or on developmental toxicity in vivo. CDNVs could be efficiently loaded with RNA and engineered to modify surface proteins. The feasibility of generating immunomodulatory CDNVs was demonstrated by preparing CDNVs with enhanced surface expression of PD1, which could bind to PD-L1 expressing tumor cells, enhance NK and T cell degranulation, and increase immune-mediated tumor cell death. These findings demonstrate the adaptability and therapeutic promise of CDNVs as promising substitutes for natural EVs that can be engineered to enhance immunomodulation.

Vermistabilization of mango tree pruning waste with five earthworm species: A biochemical and heavy metal assessment.

Mango tree pruning results in high biomass output, which is a serious agricultural and environmental problem. Vermicomposting is a potential, fast and sustainable tool to address these challenges. For sixty days, the experiment was carried out in six vermireactors containing five earthworm species by Eudrilus eugeniae, Eisenia fetida, Aporrectodea rosea, Lumbricus rubellus, and Lampito mauritii, as well as composting (without earthworm) using mango tree pruning waste biomass along with cattle dung as an instant preferred feeding material for earthworms. The pH, TOC, C/N and C/P ratios of the waste were substantially reduced by the earthworm activity. However, after vermicomposting, the levels of macronutrients (N, P, K, Ca, Mg, S) and micronutrients (Fe, Mn, Zn, and Cu) and microbial count substantially increased. The TOC content of waste was reduced by 42-55%, and the C/N of vermicompost ranged from 5.58 to 11.38. The results showed that earthworm fecundity was highest in vermireactors containing Eudrilus eugeniae and Eisenia fetida. The current study was ultimately determine that vermicomposting using Eudrilus eugeniae or Eisenia fetida is an effective strategy for utilising mango tree pruning waste, ensuring environmental sustainability and improving farmer revenue.

The Cholangiocarcinoma in the Young (CITY) Study: Tumor Biology, Treatment Patterns, and Survival Outcomes in Adolescent Young Adults With Cholangiocarcinoma.

PURPOSE: Increased awareness of the distinct tumor biology for adolescents and young adults (AYAs) with cancer has led to improvement in outcomes for this population. However, in cholangiocarcinoma (CCA), a paucity of data exist on the AYA population. To our knowledge, we present the largest study to date on AYA disease biology, treatment patterns, and survival outcomes in CCA. METHODS: A multi-institutional cohort of patients with CCA diagnosed with intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma (ECC) was used for analysis. Retrospective chart review was conducted on patients who were 50 years old and younger (young; n = 124) and older than 50 years (older; n = 723). RESULTS: Among 1,039 patients screened, 847 patients met eligibility (72% ICC, 28% ECC). Young patients had a larger median tumor size at resection compared with older patients (4.2 v 3.6 cm; P = .048), more commonly had N1 disease (65% v 43%; P = .040), and were more likely to receive adjuvant therapy (odds ratio, 4.0; 95% CI, 1.64 to 9.74). Tumors of young patients were more likely to harbor an FGFR2 fusion, BRAF mutation, or ATM mutation (P < .05 for each). Young patients were more likely to receive palliative systemic therapy (96% v 69%; P < .001), targeted therapy (23% v 8%; P < .001), and treatment on a clinical trial (31% v 19%; P = .004). Among patients who presented with advanced disease, young patients had a higher median overall survival compared with their older counterparts (17.7 v 13.5 months; 95% CI, 12.6 to 22.6 v 11.4 to 14.8; P = .049). CONCLUSION: Young patients with CCA had more advanced disease at resection, more commonly received both adjuvant and palliative therapies, and demonstrated improved survival compared with older patients. Given the low clinical trial enrollment and poor outcomes among some AYA cancer populations, data to the contrary in CCA are highly encouraging.

Grounded in reality: artificial intelligence in medical education.

Background: In a recent survey, medical students expressed eagerness to acquire competencies in the use of artificial intelligence (AI) in medicine. It is time that undergraduate medical education takes the lead in helping students develop these competencies. We propose a solution that integrates competency-driven AI instruction in medical school curriculum. Methods: We applied constructivist and backwards design principles to design online learning assignments simulating the real-world work done in the healthcare industry. Our innovative approach assumed no technical background for students, yet addressed the need for training clinicians to be ready to practice in the new digital patient care environment. This modular 4-week AI course was implemented in 2019, integrating AI with evidence-based medicine, pathology, pharmacology, tele-monitoring, quality improvement, value-based care, and patient safety. Results: This educational innovation was tested in 2 cohorts of fourth year medical students who demonstrated an improvement in knowledge with an average quiz score of 97% and in skills with an average application assignment score of 89%. Weekly reflections revealed how students learned to transition from theory to practice of AI and how these concepts might apply to their upcoming residency training programs and future medical practice. Conclusions: We present an innovative product that achieves the objective of competency-based education of students regarding the role of AI in medicine. This course can be integrated in the preclinical years with a focus on foundational knowledge, vocabulary, and concepts, and in clinical years with a focus on application of core knowledge to real-world scenarios.

Metabolic liver cancer: associations of rare and common germline variants in one-carbon metabolism and DNA methylation genes.

Animal studies implicate one-carbon metabolism and DNA methylation genes in hepatocellular carcinoma (HCC) development in the setting of metabolic perturbations. Using human samples, we investigated the associations between common and rare variants in these closely related biochemical pathways and risk for metabolic HCC development in a multicenter international study. We performed targeted exome sequencing of 64 genes among 556 metabolic HCC cases and 643 cancer-free controls with metabolic conditions. Multivariable logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs), adjusting for multiple comparisons. Gene-burden tests were used for rare variant associations. Analyses were performed in the overall sample and among non-Hispanic whites. The results show that among non-Hispanic whites, presence of rare functional variants in ABCC2 was associated with 7-fold higher risk of metabolic HCC (OR = 6.92, 95% CI: 2.38-20.15, P = 0.0004), and this association remained significant when analyses were restricted to functional rare variants observed in ≥2 participants (cases 3.2% versus controls 0.0%, P = 1.02 × 10-5). In the overall multiethnic sample, presence of rare functional variants in ABCC2 was nominally associated with metabolic HCC (OR = 3.60, 95% CI: 1.52-8.58, P = 0.004), with similar nominal association when analyses were restricted to functional rare variants observed in ≥2 participants (cases 2.9% versus controls 0.2%, P = 0.006). A common variant in PNPLA3 (rs738409[G]) was associated with higher HCC risk in the overall sample (P = 6.36 × 10-6) and in non-Hispanic whites (P = 0.0002). Our findings indicate that rare functional variants in ABCC2 are associated with susceptibility to metabolic HCC in non-Hispanic whites. PNPLA3-rs738409 is also associated with metabolic HCC risk.

Outcomes of Radiation Segmentectomy for Hepatocellular Carcinoma in Patients with Non-Alcoholic Fatty Liver Disease versus Chronic Viral Hepatitis.

Purpose: To compare the outcomes of radiation segmentectomy for early-stage hepatocellular carcinoma (HCC) in patients with non-alcoholic fatty liver disease (NAFLD) versus hepatitis C virus (HCV). Materials and Methods: A retrospective analysis of consecutive patients with NAFLD- or HCV-related HCC treated with radiation segmentectomy from 01/2017-06/2022 was performed. Eligibility criteria included solitary tumor ≤8 cm or up to 3 HCC ≤3 cm, ECOG 0-1, and absence of vascular invasion or extrahepatic spread. Imaging best response was assessed per modified Response Evaluation Criteria in Solid Tumors. Target tumor and overall progression, time-to-progression (TTP), and overall survival (OS) were calculated. All outcomes were censored for liver transplantation (LT). Complete pathologic response (CPN) was assessed in patients who underwent LT. Results: Of 142 patients included (NAFLD: 61; HCV: 81), most had cirrhosis (NAFLD: 87%; HCV: 86%) and small tumors (median size NAFLD: 2.3 cm; HCV: 2.5 cm). Patients with NAFLD had higher BMI (p<0.001) and worse ALBI scores (p=0.003). Patients with HCV were younger (p<0.001) and had higher AFP levels (p=0.034). Median radiation dose (NAFLD: 508 Gy; HCV: 452 Gy) and specific activity (NAFLD: 700 Bq; HCV: 698 Bq) were similar between cohorts. Objective response was 100% and 97% in the NAFLD and HCV cohorts, respectively. Target tumor progression occurred in 1 (2%) NAFLD and 8 (10%) HCV patients. Target tumor TTP was not met for either cohort. Overall progression occurred in 23 (38%) NAFLD and 39 (48%) HCV patients. Overall TTP was 17.4 months (95% CI 13.5-22.2) in NAFLD and 13.5 months (95% CI 0.4-26.6) in HCV patients (p=0.86). LT was performed in 27 (44%) NAFLD and 33 (41%) HCV patients, with a CPN rate of 63% and 54%, respectively. OS was not met in the NAFLD cohort and was 53.9 months (95% CI 32.1-75.7) in the HCV cohort (p=0.15). Conclusion: Although NAFLD and HCV are associated with different mechanisms of liver injury, patients with early-stage HCC treated with radiation segmentectomy achieve comparable outcomes.

Clinical trials reimagined.

Clinical trials have been a central driver of change and have provided the evidence base necessary to advance new therapies for liver diseases. This review provides a perspective on the status of trials in hepatology and a vantage point into the emerging capabilities and external forces that will shape the conduct of clinical trials in the future. The adaptations to clinical trial operations in response to the disruptions by the COVID-19 pandemic and opportunities for innovation in hepatology trials are emphasized. Future trials in hepatology will be driven by unmet therapeutic needs and fueled by technological advances incorporating digital capabilities with expanded participant-derived data collection, computing, and analytics. Their design will embrace innovative trial designs adapted to these advances and that emphasize broader and more inclusive participant engagement. Their conduct will be further shaped by evolving regulatory needs and the emergence of new stakeholders in the clinical trials ecosystem. The evolution of clinical trials will offer unique opportunities to advance new therapeutics that will ultimately improve the lives of patients with liver diseases.

Protocol for generation of multicellular spheroids through reduced gravity.

Multicellular spheroids are useful models for drug testing or studying tumor biology, but their production requires specialized approaches. Here, we present a protocol to produce viable spheroids by slow rotation around a horizontal axis using standard culture tubes. We describe steps for both seed and starter culture, and maintenance and expansion of spheroids. We detail assessment of spheroid size, count, viability, and immunohistochemistry. This protocol reduces gravitational forces that lead to cell clumping and is amenable to high-throughput use.

Utility of an Artificial Intelligence Enabled Electrocardiogram for Risk Assessment in Liver Transplant Candidates.

BACKGROUND: Post-operative cardiac complications occur infrequently but contribute to mortality after liver transplantation (LT). Artificial intelligence-based algorithms based on electrocardiogram (AI-ECG) are attractive for use during pre-operative evaluation to screen for risk of post-operative cardiac complications, but their use for this purpose is unknown. AIMS: The aim of this study was to evaluate the performance of an AI-ECG algorithm in predicting cardiac factors such as asymptomatic left ventricular systolic dysfunction or potential for developing post-operative atrial fibrillation (AF) in cohorts of patients with end-stage liver disease either undergoing evaluation for transplant or receiving a liver transplant. METHODS: A retrospective study was performed in two consecutive adult cohorts of patients who were either evaluated for LT or underwent LT at a single center between 2017 and 2019. ECG were analyzed using an AI-ECG trained to recognize patterns from a standard 12-lead ECG which could identify the presence of left ventricular systolic dysfunction (LVEF < 50%) or subsequent atrial fibrillation. RESULTS: The performance of AI-ECG in patients undergoing LT evaluation is similar to that in a general population but was lower in the presence of prolonged QTc. AI-ECG analysis on ECG in sinus rhythm had an AUROC of 0.69 for prediction of de novo post-transplant AF. Although post-transplant cardiac dysfunction occurred in only 2.3% of patients in the study cohorts, AI-ECG had an AUROC of 0.69 for prediction of subsequent low left ventricular ejection fraction. CONCLUSIONS: A positive screen for low EF or AF on AI-ECG can alert to risk of post-operative cardiac dysfunction or predict new onset atrial fibrillation after LT. The use of an AI-ECG can be a useful adjunct in persons undergoing transplant evaluation that can be readily implemented in clinical practice.

Use of a National Clinical Skills Assessment Program Improves the Clinical Competency for Correctional Nurses and Advanced Practice Providers.

The Federal Bureau of Prisons clinical skills training development (CSTD) team accomplished the planning, creation, and execution of a first-ever national clinical skills assessment program (CSAP) for nurses and advanced practice providers (APPs). Clinical skills assessment is a part of nurse and APP credentialing and privileging and must be completed for new hires along with continued biennial recredentialing accreditation standards. A training resource manual, discipline-specific skills checklist, pre-/postprogram written examination, and standard operating procedures were created. The CSTD team used commercially available manikins, food items, and easily obtainable office supplies for simulated experiential skills assessments. The CSAP provided a consistent, reproducible, and scalable approach for the orientation, assessment, and, if indicated, remediation for correctional nurses and APPs.

RAB27B Drives a Cancer Stem Cell Phenotype in NSCLC Cells Through Enhanced Extracellular Vesicle Secretion.

Cancer stem cells (CSC) within non-small cell lung carcinoma (NSCLC) tumors drive NSCLC progression, metastasis, relapse, and intrinsic chemoresistance. Understanding the mechanisms that support the malignant phenotypes of NSCLC CSCs may provide insights for improved NSCLC therapeutic interventions. Here, we report that expression of RAB27B, a small GTPase, is significantly upregulated in NSCLC CSCs when compared with bulk cancer cells (BCC). Short hairpin RNA-mediated knockdown of RAB27B leads to a loss of stem cell marker gene expression and reduced NSCLC spheroid growth, clonal expansion, transformed growth, invasion, and tumorigenicity. We find that NSCLC CSCs secrete significantly more extracellular vesicles (EV) than BCCs, and that this is RAB27B-dependent. Furthermore, CSC-derived EVs, but not BCC-derived EVs, induce spheroid growth, clonal expansion, and invasion in BCCs. Finally, RAB27B is required for CSC-derived EV-induced stemness in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating, invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Our findings further suggest that inhibition of RAB27B-dependent EV secretion may be a potential therapeutic strategy for NSCLC. Significance: Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.