Improving Reliability of Immunological Assays by Defining Minimal Criteria for Cell Fitness.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.

A systems thinking framework for understanding rising childhood obesity in the Caribbean.

OBJECTIVE: The aim of this study is to develop a systems thinking framework to describe the common complexities of childhood obesity in the Caribbean region and identify potential areas of intervention. METHODS: Group model building (GMB) is a form of systems science. Trained GMB facilitators in Puerto Rico, the US Virgin Islands, Barbados, and Trinidad and Tobago convened a group of multi-disciplinary stakeholders in a series of virtual meetings in 2021 to elaborate a hypothesis of the system driving childhood obesity represented by causal loop diagrams (CLD). Commonalities and differences between the CLDs from each island were identified and reconciled to create a synthesized CLD. RESULTS: A single explanatory CLD across the islands was developed and includes nine reinforcing loops. These loops addressed the interconnected role of schools, policy, commercial determinants, community and the personal experience of the child in rising childhood obesity rates. CONCLUSIONS: Despite differences across settings, there is a core system driving childhood obesity in the Caribbean, as described by stakeholders in GMB workshops. Policy solutions to the problem must be multi-faceted and multi-level to address the interlinked reinforcing loops of the complex system and reduce rates of childhood obesity.

Croton stipulaceus Kunth, a native Mexican medicinal plant with antioxidant and anti-inflammatory activities / Croton stipulaceus Kunth, planta medicinal mexicana con actividad antioxidante y antiinflamatoria

Leaves of Croton stipulaceuswere extracted (EHex, ECHCl3and EEtOH extracts) to assesstheir antioxidant potential, anti-inflammatory activity in murine models and acute toxicity. EEtOH showed the highest effect in DPPH (37.80% inhibition), FRAP (1065.00 ± 55.30 µmolFe2+) and total polyphenols (231.24 ± 9.05 meq AG/gM). EHex was the most active, ~ 50% inhibition of TPA-induced ear edema; while EEtOH (dose of 2 mg/ear) showed the highest inhibition in the chronic model (97% inhibition), and inhibited MPO activity (48%). In carrageenan-induced edema, ECHCl3(dose 500 mg/kg) was the most active. None of the extracts showed acute toxicity (LD50) at 2 g/kg (p.o.). This work is the first report that supports the traditional use of C. stipulaceusas an anti-inflammatory.

De las hojas de Croton stipulaceusse obtuvieron diferentes extractos (EHex, ECHCl3y EEtOH) evaluando el potencial antioxidante y la actividad antiinflamatoria en modelos murinos y la toxicidad aguda. El EEtOH mostró mayor efecto en DPPH (37.80% inhibición), FRAP (1065.00 ± 55.30 µmolFe2+) y polifenolestotales (231.24 ± 9.05 meq AG/gM). El EHex fue el más activo, cercano al 50% de inhibición del edema auricular inducido con TPA; mientras que el EEtOH (dosis de 2 mg/oreja) mostró la mayor inhibición en el modelo crónico (97% inhibición), e inhibió la actividad de la MPO (48%). En el edema inducido con carragenina, el ECHCl3(dosis 500 mg/kg) fue el más activo. Ninguno de los extractos mostró una toxicidad aguda (DL50) mayor a 2 g/kg (p.o). Este trabajo es el primer reporte que sustenta el uso tradicional de C. stipulaceuscomo antiinflamatorio.

Distribution and Endemism Areas of Bonamia Thouars (Convolvulacea) in Brazil.

Brazil harbors the highest richness of Convolvulaceae with 424 species recognized mainly distributed in the Amazon, Atlantic Forest, Caatinga and Cerrado phytogeographic domains. Seventeen of these species are representatives of Bonamia, with ten endemic to the country. The aim of the study was to map the distribution of this group to understand its richness, its sampling and detecting areas of endemism, valuable information for conservation. We collected data gathered from herbaria and from the online database. The data were refined (1) excluding of records not at the species level; (2) records with no identification of collection site or with only the identification of the state of collection. There was calculated the richness, the number of records and an estimate of richness per cell. We conducted a parsimony analysis of endemism for distribution analysis. Finally, the knowledge of richness for the species was analyzed. There were gathered 420 occurrence records, in 87 grid cells. Most grid cells observed in the study presented one species. Two endemic areas were found for the genus. The results contribute to the understanding of the distribution of the group in Brazil, highlighting shortfalls in collections.

Understanding the Saffron Corm Development-Insights into Histological and Metabolic Aspects.

The reproduction of Crocus sativus L., a sterile triploid plant, is carried out exclusively through corms, whose size determines the saffron yield. The development of daughter corms (DC) is supported by photoassimilates supplied by the leaves as well as by the mother corms (MC). While biomass partitioning during DC development is well studied, growth dynamics in terms of cell number and size, the involved meristems, as well as carbohydrate partition and allocation, are not yet fully understood. We conducted a comprehensive study into saffron corm growth dynamics at the macroscopic and microscopic levels. Variations in carbohydrate content and enzymatic activities related to sucrose metabolism in sources and sinks were measured. Two key meristems were identified. One is involved in vascular connections between DC and MC. The other is a thickening meristem responsible for DC enlargement. This research explains how the previously described phases of corm growth correlate with variations in cell division, enlargement dynamics, and carbohydrate partitioning among organs. Results also elucidated that the end of DC growth relates to a significant drop in MC root biomass, limiting the water supply for the DC growth, and establishing the onset of leaf wilting. The lack of starch accumulation in aged leaf cells is noteworthy, as is the accumulation of lipids. We hypothesize a signaling role of sugars in DC growth initiation, stop, and leaf aging. Finally, we established a predominant role of sucrose synthase as a sucrolytic enzyme in the maintenance of the high flux of carbon for starch synthesis in DC. Together, the obtained results pave the way for the definition of strategies leading to better control of saffron corm development.

"Doing well": Intraoperative entrustable professional activity assessments provided limited technical feedback.

Background: Entrustable Professional Activities (EPAs) allow for the assessment of specific, observable, essential tasks in medical education. Since being developed in non-surgical fields, EPA assessments have been implemented in surgery to explore intraoperative entrustment. However, assessment burden is a significant problem for faculty, and it is unknown whether EPA assessments enable formative technical feedback. EPAs' formative utility could inform how surgical programs facilitate technical feedback for trainees. We aimed to assess the extent to which narrative comments provided through the Fellowship Council (FC) EPA assessments contained technical feedback. Methods: The FC previously collected EPA assessments for subspecialty surgical fellows from September 2020 to October 2022. Two raters reviewed assessments' narrative comments for inclusion of each skill area that makes up part of the Objective Structured Assessment of Technical Skills (OSATS). A third rater reconciled discrepant ratings. Results: During the study period, there were 3302 completed EPA assessments, including 1191 fellow self-assessments, 1124 faculty assessments, and 987 assessments without an identified assessor role. We found that assessments' narrative comments related to a median of two of the seven OSATS areas (IQR:1-2). There were no comments relevant to any of the seven OSATS areas in 16.0 % of all assessments. Conclusions: In this review of narrative comments for EPA assessments from the FC, we found that limited technical feedback of the kind included in the OSATS was provided in many assessments. These results suggest benefit to adjusting the EPA form, enhancing faculty development, or continuing additional types of targeted technical assessment intraoperatively. Key message: This analysis of narrative comments from fellowship EPA assessments showed that many assessments included limited technical feedback. To allow for continued technical feedback for fellows, these results highlight the need for further refinements of the EPA assessment form, additional faculty development, or ongoing use of other types of technical assessment.

Association of Variability and Hypertensive Loads in 24-h Blood Pressure With Mortality and Cardiovascular Risk.

BACKGROUND: Evidence shows that high 24-h blood pressure (BP) variability increases cardiovascular risk. We investigated whether 24-h BP variability relates to mortality and cardiovascular risk due to inherent variability and/or hypertensive loads in 24-h BP. METHODS: A total of 1,050 participants from the Maracaibo Aging Study (mean age, 66 years; women, 67.2%) underwent 24-h ambulatory BP monitoring and were followed between 2001 and 2016. To evaluate inherent BP variability, we used average real variability (ARV) as it captures variability among consecutive BP readings. 24-h systolic BP load was the proportion (%) of systolic BP readings ≥130 mm Hg during the daytime and ≥110 during the nighttime. Our primary endpoint was total mortality and major adverse cardiovascular endpoints (MACE). Statistics included Cox proportional models. RESULTS: During a median follow-up of 8.3 years, 299 participants died and 210 experienced MACE. Each +2 mm Hg (corresponding to 1-standard deviation) higher 24-h systolic ARV (mean value, 9.0 ±â€…2.0 mm Hg) was associated with higher hazard ratios (HRs) for mortality by 1.28-fold (95% confidence interval [CI], 1.14-1.43) and for MACE by 1.24-fold (95% CI, 1.08-1.42). Each 30% higher 24-h systolic BP load (median value, 63%) was associated with mortality and MACE with HRs of 1.29 (95% CI, 1.15-1.46) and 1.28 (95% CI, 1.10-1.48); respectively. After models were additionally adjusted by BP level, only ARV was associated with mortality (HR, 1.17; 95% CI, 1.04-1.33) and MACE (HR, 1.16; 95% CI, 1.00-1.34). CONCLUSIONS: High ARV and hypertensive loads in 24-h systolic BP were associated with mortality and cardiovascular risk, however, only ARV is associated independently of the BP level.

Flaxseed Oil (Linum usitatissimum) Prevents Cognitive and Motor Damage in Rats with Hyperammonemia.

Hyperammonemia is characterized by the excessive accumulation of ammonia in the body as a result of the loss of liver detoxification, leading to the development of hepatic encephalopathy (HE). These metabolic alterations carry cognitive and motor deficits and cause neuronal damage, with no effective treatment at present. In this study, we aimed to evaluate the effect of two subacute oral administrations of flaxseed oil (0.26 and 0.52 mL/kg) on short- and long-term memory, visuospatial memory, locomotor activity, motor coordination, and the neuronal morphology of the prefrontal cortex (PFC) via tests on Wistar rats with hyperammonemia. The goal was to identify its role in the regulation of cerebral edema, without liver damage causing cerebral failure. In contrast with an ammonium-rich diet, flaxseed oil and normal foods did not cause cognitive impairment or motor alterations, as evidenced in the short-term and visuospatial memory tests. Furthermore, the flaxseed oil treatment maintained a regular neuronal morphology of the prefrontal cortex, which represents a neuroprotective effect. We conclude that the oral administration of flaxseed oil prevents cognitive and motor impairments as well as neuronal alterations in rats with hyperammonemia, which supports the potential use of this oil to ameliorate the changes that occur in hepatic encephalopathy.

Designing Calcium Silicate Cements with On-Demand Properties for Precision Endodontics.

Calcium silicate (C3S) cements are available in kits that do not account for patients' specific needs or clinicians' preferences regarding setting time, radiopacity, mechanical, and handling properties. Moreover, slight variations in powder components and liquid content affect cement's properties and bioactivity. Unfortunately, it is virtually impossible to optimize several cement properties simultaneously via the traditional "one variable at a time" strategy, as inputs often induce trade-offs in properties (e.g., a higher water-to-powder ratio [W/P] increases flowability but decreases mechanical properties). Herein, we used Taguchi's methods and genetic algorithms (GAs) to simultaneously analyze the effect of multiple inputs (e.g., powder composition, radiopacifier concentration, and W/P) on setting time, pH, flowability, diametral tensile strength, and radiopacity, as well as prescribe recipes to produce cements with predicted properties. The properties of cements designed with GAs were experimentally tested, and the results matched the predictions. Finally, we show that the cements increased the genetic expression of odonto/osteogenic genes, alkaline phosphatase activity, and mineralization potential of dental pulp stem cells. Hence, GAs can produce cements with tailor-made properties and differentiation potential for personalized endodontic treatment.

Associations between the Gut Microbiota, Race, and Ethnicity of Patients with Colorectal Cancer: A Pilot and Feasibility Study.

BACKGROUND: Colorectal cancer (CRC) is more prevalent among some racial and ethnic minority and low socioeconomic status populations. Although the gut microbiota is a risk factor for CRC and varies with race and ethnicity, its role in CRC disparities remains poorly understood. METHODS: We examined the feasibility of recruiting sociodemographically diverse CRC patients for a microbiome study involving a home stool collection. We also explored whether race and ethnicity were associated with gut microbiome composition. We recruited Black/African American, Hispanic/Latino, and non-Hispanic White patients who were receiving care for active CRC to complete a comprehensive dietary and lifestyle survey, self-collect a stool sample, and complete an exit interview. Gut microbial diversity and composition were analyzed using 16S rRNA gene sequencing. RESULTS: 30 individuals consented (of 35 who were eligible and contacted) with 5 (17%) Black/African American, 11 (37%) Hispanic/Latino, and 14 (46%) non-Hispanic White. A total of 22 (73%) completed the dietary and lifestyle survey; 18 (63%) returned a stool sample. Even after controlling for socioeconomic, dietary, or treatment-related covariates, microbiome composition was associated with race and ethnicity. Fusobacteriota (a phylum associated with the development and progression of CRC) was significantly higher in the Black/African American group compared to others, and microbial diversity was higher in samples from non-Hispanic White individuals compared to Hispanic/Latino individuals. CONCLUSION: Our study shows that it is feasible to recruit and collect stool samples from diverse individuals with CRC and found significant associations in gut microbial structure with race and ethnicity.

Five new species of Gloeandromyces (Fungi, Laboulbeniales) from tropical American bat flies (Diptera, Streblidae), revealed by morphology and phylogenetic reconstruction.

This paper describes and illustrates five new species of Gloeandromyces (Ascomycota, Laboulbeniales) associated with tropical American bat flies (Diptera, Streblidae). These are Gloeandromyces cusucoensis sp. nov. from Trichobius uniformis in Costa Rica and Honduras, G. diversiformis sp. nov. from Strebla wiedemanni in Costa Rica, G. plesiosaurus sp. nov. from Trichobius yunkeri in Panama, G. pseudodickii sp. nov. from Trichobius longipes in Ecuador and Panama, and G. verbekeniae sp. nov. from Strebla galindoi in Ecuador and Panama. The description of these five species doubles the number of known species in the genus. Morphological characteristics, host association, and a three-locus (18S nuc rDNA, 28S nuc rDNA, TEF1) phylogenetic reconstruction support placement of these taxa in the genus Gloeandromyces. Three of the new species are polymorphic; they have multiple morphotypes that grow in specific positions on the host integument: G. diversiformis f. diversiformis, f. musiformis, and f. vanillicarpiformis; G. plesiosaurus f. asymmetricus and f. plesiosaurus; and G. verbekeniae f. verbekeniae and f. inflexus. Finally, a dichotomous key to all species and morphotypes is presented.

An ECD and NMR/DP4+ Computational Pipeline for Structure Revision and Elucidation of Diphenazine-Based Natural Products.

Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein-protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.

Incidence of interstitial lung disease and cardiotoxicity with trastuzumab deruxtecan in breast cancer patients: a systematic review and single-arm meta-analysis.

BACKGROUND: Trastuzumab deruxtecan (T-DXd) has been shown to benefit progression-free survival and overall survival in patients with metastatic breast cancer (mBC) after progression on ≥1 human epidermal growth factor receptor 2 (HER2)-targeted therapies. However, interstitial lung disease (ILD) and cardiotoxicity are the most significant toxicities associated with T-DXd. Therefore, we conducted a systematic review and meta-analysis to assess the incidence and severity of these toxicities in mBC patients treated with T-DXd. MATERIALS AND METHODS: We searched PubMed, Cochrane, and Scopus databases, and conferences websites for randomized clinical trials and nonrandomized studies of intervention including HER2-low or HER2-positive mBC patients who received at least one dose of T-DXd. Statistical analysis was carried out using R software. RESULTS: We included 15 studies comprising 1970 patients with a mean follow-up of 13.3 months. Median age ranged from 53 to 59 years, 61.9% were non-Asian, and 67.4% had hormone receptor-positive mBC. In a pooled analysis, the incidence of ILD was 11.7% [222 patients; 95% confidence interval (CI) 9.1% to 15.0%]. Patients receiving T-DXd dose of 6.4 mg/kg developed a significantly higher rate of ILD (22.7%) compared to those receiving a dose of 5.4 mg/kg (9.3%) (P < 0.01). Most cases of ILD (80.2%; 174/217 patients) were mild (grade 1 or 2). Grade 3 or 4 ILD was reported in 29 patients (13.4%), and grade 5 in 14 patients (6.4%). The incidence of decreased left ventricular ejection fraction (LVEF) was 1.95% (95% CI 0.65% to 3.73%), and the QT interval (QTi) prolongation was 7.77% (95% CI 2.74% to 20.11%). Most patients were asymptomatic, but four had LV dysfunction and heart failure (0.26%). CONCLUSIONS: In this meta-analysis of 1970 patients with mBC, treatment with T-DXd was associated with a 11.7% incidence of ILD, 7.7% incidence of prolonged QTi, and 1.9% incidence of reduced LVEF. Early detection and management of T-DXd-related toxicity by a multidisciplinary team may ultimately improve patient outcomes.

Ectopic expression of the AtCDF1 transcription factor in potato enhances tuber starch and amino acid contents and yield under open field conditions.

Introduction: Cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. Methods: In this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. Results: In growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. Discussion: The results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants.

Impact of a Comprehensive Financial Resource on Financial Toxicity in a National, Multiethnic Sample of Adult, Adolescent/Young Adult, and Pediatric Patients With Cancer.

PURPOSE: Financial toxicity is a well-recognized problem for patients with cancer. However, a crucial gap remains in describing and implementing mitigation strategies. We conducted a national survey of a multiethnic adolescent/pediatric and adult patient population served by Family Reach, a nonprofit organization focused on removing financial barriers to cancer care, to evaluate the impact of a comprehensive financial resource on patient-reported financial toxicity. METHODS: An electronic survey was administered to characterize patients' current financial health and the impact of Family Reach's resources on financial toxicity. The survey was e-mailed to all patients or caregivers who received resources from Family Reach between January 1, 2020, and June 30, 2020. Factors associated with higher financial stress and higher potential impact of resources on financial burden were evaluated through separate multivariate regression models. Qualitative responses were analyzed using manual coding and thematic analysis. RESULTS: Three hundred thirty socioeconomically and racially diverse respondents (overall response rate 40%; 46% non-Hispanic White; 48% with incomes below the federal poverty line) completed the survey and were included in the analysis. More than half of respondents reported high financial stress in the previous week. Hispanic ethnicity, Black race, and low annual household income were associated with higher financial toxicity. A greater amount of financial assistance was associated with a higher confidence rating that resources provided would decrease financial stress. In open-ended comments, respondents highlighted the impact of the COVID-19 pandemic and resulting job loss on financial toxicity, the importance of financial navigation, the benefits of financial assistance, and anxiety about long-term financial health. CONCLUSION: A comprehensive financial resource, particularly financial assistance, alleviated financial toxicity in a multiethnic national sample of patients with cancer. Ongoing work is critical to address sustainable funding sources and financial navigation to support patients during treatment and survivorship.

AHPBA graduated fellows' assessments of strengths, weaknesses, opportunities and threats facing fellowship training in HPB surgery: "HPB training excellence means volume, knowledge, and mentorship".

BACKGROUND: Many fellowship programs in North America prepare surgeons for a career in Hepato-Pancreato-Biliary (HPB) surgery. Recent fellowship graduates were surveyed as part of a strengths, weaknesses, opportunities, and threats (SWOT) analysis commissioned by Americas Hepato-Pancreato-Biliary Association (AHPBA). METHODS: This was a cross-sectional study surveying AHPBA-certified fellowship graduates conducted August-December 2021. Survey data were analyzed using descriptive statistics. Free-text answers were analyzed using both grounded theory principles and thematic network analyses. RESULTS: Four main themes were identified: (i) concerns regarding the lack of standardization between HPB fellowship curricula (ii) concern for job market oversaturation, (iii) need to emphasize the value in HPB fellowship training and (iv) importance of diversity, inclusion, and equity in HPB training. DISCUSSION: Based on themes identified, the strengths of AHPBA-certified HPB programs include superior case volume and technical training. Areas of weakness and growth opportunities include standardizing training experiences. According to AHPBA-certificate awardees, optimizing future HPB fellowships would include strong sponsorship for job placement after graduation, and more intentional investments in diversity, equity, and inclusion.

AHPBA senior leaders' assessments of strengths, weaknesses, opportunities, and threats facing fellowship training in HPB surgery: "We need to standardize our training experiences".

BACKGROUND: Multiple fellowship programs in North America prepare surgeons for a career in Hepato-Pancreatico-Biliary (HPB) surgery. Inconsistent operative experiences and disease process exposures across programs and pathways produces variability in training product and therefore, lack of clarity around what trained HPB surgeons are prepared to do in early practice. Thus, a strengths, weaknesses, opportunities, and threats (SWOT) analysis of AHPBA fellowship training was conducted. METHODS: This was a mixed-methods, cross-sectional study. Eleven AHPBA-Founding Members (FM) and 24 current or former Program Directors (PD) of programs eligible for AHPBA certificates were surveyed and interviewed. Grounded theory principles and thematic network analysis were used to analyze interview transcripts. Descriptive statistics were used to analyze survey data. RESULTS: Three main themes were identified: (i) Concern for training rigor and consistency (ii) Desire to standardize curricula and broaden training requirements and, (iii) Need to validate both the value of training and job marketability via certification. DISCUSSION: Based on the themes identified, the strengths of AHPBA-certified HPB programs include superior technical training and case volumes. Areas of improvement included elevating baseline competencies by increasing required case volume and breadth to ensure minimally invasive experience, operative autonomy, and multidisciplinary care coordination.