OprF Impacts Pseudomonas aeruginosa Biofilm Matrix eDNA Levels in a Nutrient-Dependent Manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm are limited. Here, we identify a nutrient-dependent effect of OprF in static biofilms, whereby ΔoprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa ΔoprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here, we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A ΔoprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective ΔoprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in biofilms.

Accurate Identification of Mucinous Pancreatic Cystic Lesions Using Small-Volume Analytes.

INTRODUCTION: The accurate identification of mucinous pancreatic cystic lesions (PCLs) is paramount for cancer risk stratification. Cyst fluid carcinoembryonic antigen (CEA), the only routinely used test, requires high volumes and has low sensitivity. We aimed to compare the performance of two investigational small-volume biomarkers, glucose and the protease gastricsin, to CEA for PCL classification. METHODS: We obtained cyst fluid samples from 81 patients with pathologically confirmed PCLs from four institutions between 2003 and 2016. Gastricsin activity was measured using an internally quenched fluorescent substrate. Glucose levels were measured with a standard glucometer. CEA levels were obtained from the medical record. Models using Classification and Regression Trees were created to predict mucinous status. Model performance was evaluated using nested cross-validation. RESULTS: Gastricsin activity, CEA, and glucose levels from patients with mucinous (n = 50) and nonmucinous (n = 31) PCLs were analyzed. Area under the curve (AUC) was similar for individual classifiers (gastricsin volume normalized [GVN] 0.88; gastricsin protein concentration normalized [GPN] 0.95; glucose 0.83; CEA 0.84). The combination of two classifiers did not significantly improve AUC, with CEA + GVN (0.88) performing similarly to CEA + GPN (0.95), GVN + glucose (0.87), GPN + glucose (0.95), and CEA + glucose (0.84). The three-analyte combination performed similarly to single and dual classifiers (GPN + glucose + CEA AUC 0.95; GVN + glucose + CEA AUC 0.87). After multiple comparison corrections, there were no significant differences between the individual, dual, and triple classifiers. CONCLUSIONS: Gastricsin and glucose performed similarly to CEA and required <5% of the volume required for CEA; these classifiers may be useful in patients with limited cyst fluid. Future multicenter prospective studies are needed to validate and compare these novel small-volume biomarkers.

EFEMP1 rare variants cause familial juvenile-onset open-angle glaucoma.

Juvenile open-angle glaucoma (JOAG) is a severe type of glaucoma with onset before age 40 and dominant inheritance. Using exome sequencing we identified 3 independent families from the Philippines with novel EFEMP1 variants (c.238A>T, p.Asn80Tyr; c.1480T>C, p.Ter494Glnext*29; and c.1429C>T, p.Arg477Cys) co-segregating with disease. Affected variant carriers (N = 34) exhibited severe disease with average age of onset of 16 years and with 76% developing blindness. To investigate functional effects, we transfected COS7 cells with vectors expressing the three novel EFEMP1 variants and showed that all three variants found in JOAG patients caused significant intracellular protein aggregation and retention compared to wild type and also compared to EFEMP1 variants associated with other ocular phenotypes including an early-onset form of macular degeneration, Malattia Leventinese/Doyne's Honeycomb retinal dystrophy. These results suggest that rare EFEMP1 coding variants can cause JOAG through a mechanism involving protein aggregation and retention, and that the extent of intracellular retention correlates with disease phenotype. This is the first report of EFEMP1 variants causing JOAG, expanding the EFEMP1 disease spectrum. Our results suggest that EFEMP1 mutations appear to be a relatively common cause of JOAG in Filipino families, an ethnically diverse population.

Evidence-based Guidelines on the Use of Virtual Surgical Education Pertaining to the Domains of Cognition and Curriculum, Psychomotor Skills Training, and Faculty Development and Mentorship.

OBJECTIVE: To identify, categorize, and evaluate the quality of literature, and to provide evidence-based guidelines on virtual surgical education within the cognitive and curricula, psychomotor, and faculty development and mentorship domains. SUMMARY OF BACKGROUND DATA: During the coronavirus disease 2019 pandemic, utilizing virtual learning modalities is expanding rapidly. Although the innovative methods must be considered to bridge the surgical education gap, a framework is needed to avoid expansion of virtual education without proper supporting evidence in some areas. METHODS: The Association for Surgical Education formed an ad-hoc research group to evaluate the quality and methodology of the current literature on virtual education and to build evidence-based guidelines by utilizing the SiGN methodology. We identified patient/problem-intervention-comparison-outcome-style questions, conducted systematic literature reviews using PubMed, EMBASE, and Education Resources information Center databases. Then we formulated evidence-based recommendations, assessed the quality of evidence using Grading of Recommendations, Assessment, Development, and Evaluation, Newcastle-Ottawa Scale for Education, and Kirkpatrick ratings, and conducted Delphi consensus to validate the recommendations. RESULTS: Eleven patient/problem-intervention-comparison-outcome-style questions were designed by the expert committees. After screening 4723 articles by the review committee, 241 articles met inclusion criteria for full article reviews, and 166 studies were included and categorized into 3 domains: cognition and curricula (n = 92), psychomotor, (n = 119), and faculty development and mentorship (n = 119). Sixteen evidence-based recommendations were formulated and validated by an external expert panel. CONCLUSION: The evidence-based guidelines developed using SiGN methodology, provide a set of recommendations for surgical training societies, training programs, and educators on utilizing virtual surgical education and highlights the area of needs for further investigation.

CRISPR/Cas9 and genetic screens in malaria parasites: small genomes, big impact.

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes.

The ACMG SF v3.0 gene list increases returnable variant detection by 22% when compared with v2.0 in the ClinSeq cohort.

PURPOSE: The American College of Medical Genetics and Genomics (ACMG) recommends the return of pathogenic and likely pathogenic (P/LP) secondary findings from exome and genome sequencing. The latest version (ACMG secondary finding [SF] v3.0) includes 14 additional genes. We interrogated the ClinSeq cohort for variants in these genes to determine the additional yield in unselected individuals. METHODS: Exome data from 1473 individuals (60% White, 34% African American or Black, 6% other) were analyzed. We restricted our analyses to coding variants; +1,+2,-1, and -2 splice site variants; and the pathogenic GAA variant, NM_000152.5:c.-32-13T>G. Variants were assessed with slightly modified ACMG/Association of Molecular Pathology guidelines. RESULTS: A total of 25 P/LP variants were identified. In total, 7 individuals had P/LP variants in genes recommended for return of heterozygous variants, namely HNF1A (1), PALB2 (3), TMEM127 (1), and TTN (2). In total, 4 individuals had a homozygous variant in a gene recommended for biallelic variant return, namely HFE, NM_000410.3(HFE):c.845G>A p.Cys282Tyr. A total of 17 P/LP variants were identified in the heterozygous state in genes recommended only for biallelic variant reporting and were not returned. The frequency of returnable P/LP variants did not significantly differ by race. CONCLUSION: Using the ACMG SF v3.0, the returnable P/LP variant frequency increased in the ClinSeq cohort by 22%, from 3.4% (n = 50, ACMG SF v2.0) to 4.1% (n = 61, ACMG SF v3.0).

Association of Race/Ethnicity With Substance Use Testing After Trauma: A Cross-Sectional Study.

INTRODUCTION: Race/ethnicity has been strongly associated with substance use testing but little is known about this association in injured patients. We sought to identify trends and associations between race/ethnicity and urine toxicology (UTox) or Blood Alcohol Concentration (BAC) testing in a diverse population after trauma. MATERIALS AND METHODS: We conducted a retrospective cross-sectional study of adult trauma patients admitted to a single Level-1 trauma center from 2012 to 2019. The prevalence of substance use testing was evaluated over time and analyzed using a multivariable logistic regression, with a subgroup analysis to evaluate the interaction of English language proficiency with race/ethnicity in the association of substance use testing. RESULTS: A total of 15,556 patients (40% White, 13% Black, 24% Latinx, 20% Asian, and 3% Native or Unknown) were included. BAC testing was done in 63.2% of all patients and UTox testing was done in 39.2%. The prevalence of substance use testing increased over time across all racial/ethnic groups. After adjustment, Latinx patients had higher odds of receiving a BAC test and Black patients had higher odds of receiving a UTox test (P < 0.001 and P < 0.001, respectively) compared to White patients. Asian patients had decreased odds of undergoing a UTox or BAC test compared to White patients (P < 0.001 and P < 0.001, respectively). Patients with English proficiency had higher odds of undergoing substance use testing compared to those with limited English proficiency (P < 0.001). CONCLUSIONS: Despite an increase in substance use testing over time, inequitable testing remained among racial/ethnic minorities. More work is needed to combat racial/ethnic disparities in substance use testing.

The Association of Limited English Proficiency With Morbidity and Mortality After Trauma.

INTRODUCTION: Disparities following traumatic injury by race/ethnicity and insurance status are well-documented. However, the relationship between limited English proficiency (LEP) and outcomes after trauma is poorly understood. This study describes the association between LEP and morbidity and mortality after traumatic injury. METHODS: A retrospective cohort study was conducted of adult trauma patients admitted to a level 1 trauma center from 2012 to 2018. Morbidity (length of stay [LOS], intensive care unit admission, intensive care unit LOS, discharge destination) and in-hospital mortality for LEP and English proficient (EP) patients were compared using univariate and multivariable logistic and generalized linear models controlling for patient demographics (age, sex, race/ethnicity, insurance) and clinical characteristics (mechanism, activation level, Glasgow Coma Scale, Injury Severity Score, traumatic brain injury). RESULTS: Of the 13,104 patients, 16% were LEP patients. LEP languages included Chinese (44%) and Spanish (38%), and 18% categorized as "Other," including 33 languages. In multivariable models, LEP was statistically significantly associated with increased hospital LOS (P = 0.003) and increased discharge to home with home health services (P = 0.042) or to skilled nursing facility/rehabilitation (P = 0.006). Mortality rate was 7% for LEP versus 4% for EP patients (P < 0.0001). In multivariable analysis, speaking an LEP language other than Chinese or Spanish was statistically significantly associated with increased mortality compared to EP (P = 0.006). CONCLUSIONS: Following traumatic injury, LEP patients experience increased hospital LOS and are more frequently discharged to home with home health services or to skilled nursing facilities/rehabilitation. LEP patients speaking languages other than Chinese or Spanish experience increased mortality compared to EP patients.

Comparison of advanced techniques for local excision of rectal lesions: a case series.

BACKGROUND: Robotic transanal minimally invasive surgery (R-TAMIS) is an appealing alternative to transanal minimally invasive surgery (TAMIS) and transanal endoscopic microsurgery (TEM) for benign and early malignant rectal lesions that are not amenable to traditional open transanal excision. However, no studies to our knowledge have directly compared the three techniques. This study sought to compare peri-operative and pathologic outcomes of the three approaches. METHODS: The records of 29 consecutive patients who underwent TEM, TAMIS, or R-TAMIS at a single academic center between 2016 and 2020 were reviewed. Intra-operative details, pathological diagnosis and margins, and post-operative outcomes were recorded. The three groups were compared using chi-square and Kruskal-Wallis tests. RESULTS: Overall, 16/29 patients were women and the median age was 57 (interquartile range (IQR): 28-81). Thirteen patients underwent TEM, six had TAMIS, and 10 had R-TAMIS. BMI was lower in the R-TAMIS patients (24.7; IQR 23.8-28.7), than in TEM (29.3; IQR 19.9-30.2), and TAMIS (30.4; IQR 26.6-32.9) patients. High grade dysplasia and/or invasive cancer was more common in TAMIS (80%) and R-TAMIS (66.7%) patients than in TEM patients (41.7%). The three groups did not differ significantly in tumor type or distance from the anal verge. No R-TAMIS patients had a positive surgical margin compared to 23.1% in the TEM group and 16.7% in the TAMIS group. Length of stay (median 1 day for TEM and R-TAMIS patients, 0 days for TAMIS patients) and 30-day readmission rates (7.7% of TEM, 0% of TAMIS, 10% of R-TAMIS patients) also did not differ among the groups. Median operative time was 110 min for TEM, 105 min for TAMIS, and 76 min for R-TAMIS patients. CONCLUSIONS: R-TAMIS may have several advantages over other advanced techniques for transanal excisions. R-TAMIS tended to be faster and to more often result in negative surgical margins compared to the two other techniques.

Patient Perceptions on Barriers and Facilitators to Accessing Low-acuity Surgery During COVID-19 Pandemic.

BACKGROUND: The onset of the COVID-19 pandemic led to the postponement of low-acuity surgical procedures in an effort to conserve resources and ensure patient safety. This study aimed to characterize patient-reported concerns about undergoing surgical procedures during the pandemic. METHODS: We administered a cross-sectional survey to patients who had their general and plastic surgical procedures postponed at the onset of the pandemic, asking about barriers to accessing surgical care. Questions addressed dependent care, transportation, employment and insurance status, as well as perceptions of and concerns about COVID-19. Mixed methods and inductive thematic analyses were conducted. RESULTS: One hundred thirty-five patients were interviewed. We identified the following patient concerns: contracting COVID-19 in the hospital (46%), being alone during hospitalization (40%), facing financial stressors (29%), organizing transportation (28%), experiencing changes to health insurance coverage (25%), and arranging care for dependents (18%). Nonwhite participants were 5 and 2.5 times more likely to have concerns about childcare and transportation, respectively. Perceptions of decreased hospital safety and the consequences of possible COVID-19 infection led to delay in rescheduling. Education about safety measures and communication about scheduling partially mitigated concerns about COVID-19. However, uncertainty about timeline for rescheduling and resolution of the pandemic contributed to ongoing concerns. CONCLUSIONS: Providing effective surgical care during this unprecedented time requires both awareness of societal shifts impacting surgical patients and system-level change to address new barriers to care. Eliciting patients' perspectives, adapting processes to address potential barriers, and effectively educating patients about institutional measures to minimize in-hospital transmission of COVID-19 should be integrated into surgical care.

In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

Patterns of pharmacogenetic variation in nine biogeographic groups.

Frequencies of pharmacogenetic (PGx) variants are known to differ substantially across populations but much of the available PGx literature focuses on one or a few population groups, often defined in nonstandardized ways, or on a specific gene or variant. Guidelines produced by the Clinical Pharmacogenetic Implementation Consortium (CPIC) provide consistent methods of literature extraction, curation, and reporting, including comprehensive curation of allele frequency data across nine defined "biogeographic groups" from the PGx literature. We extracted data from 23 CPIC guidelines encompassing 19 genes to compare the sizes of the populations from each group and allele frequencies of altered function alleles across groups. The European group was the largest in the curated literature for 16 of the 19 genes, while the American and Oceanian groups were the smallest. Nearly 200 alleles were detected in nonreference groups that were not reported in the largest (reference) group. The genes CYP2B6 and CYP2C9 were more likely to have higher frequencies of altered function alleles in nonreference groups compared to the reference group, while the genes CYP4F2, DPYD, SLCO1B1, and UGT1A1 were less likely to have higher frequencies in nonreference groups. PGx allele frequencies and function differ substantially across nine biogeographic groups, all but two of which are underrepresented in available PGx data. Awareness of these differences and increased efforts to characterize the breadth of global PGx variation are needed to ensure that implementation of PGx-guided drug selection does not further widen existing health disparities among populations currently underrepresented in PGx data.

Massive contractions of myotonic dystrophy type 2-associated CCTG tetranucleotide repeats occur via double-strand break repair with distinct requirements for DNA helicases.

Myotonic dystrophy type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75 to 11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables the selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are involved in these massive contractions, indicating a mechanism that uses homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a short-track control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

Design and Characterization of Model Systems that Promote and Disrupt Transparency of Vertebrate Crystallins In Vitro.

Positioned within the eye, the lens supports vision by transmitting and focusing light onto the retina. As an adaptive glassy material, the lens is constituted primarily by densely-packed, polydisperse crystallin proteins that organize to resist aggregation and crystallization at high volume fractions, yet the details of how crystallins coordinate with one another to template and maintain this transparent microstructure remain unclear. The role of individual crystallin subtypes (α, ß, and γ) and paired subtype compositions, including how they experience and resist crowding-induced turbidity in solution, is explored using combinations of spectrophotometry, hard-sphere simulations, and surface pressure measurements. After assaying crystallin combinations, ß-crystallins emerged as a principal component in all mixtures that enabled dense fluid-like packing and short-range order necessary for transparency. These findings helped inform the design of lens-like hydrogel systems, which are used to monitor and manipulate the loss of transparency under different crowding conditions. When taken together, the findings illustrate the design and characterization of adaptive materials made from lens proteins that can be used to better understand mechanisms regulating transparency.

Massive contractions of Myotonic Dystrophy Type 2-associated CCTG tetranucleotide repeats occur via double strand break repair with distinct requirements for helicases.

Myotonic Dystrophy Type 2 (DM2) is a genetic disease caused by expanded CCTG DNA repeats in the first intron of CNBP. The number of CCTG repeats in DM2 patients ranges from 75-11,000, yet little is known about the molecular mechanisms responsible for repeat expansions or contractions. We developed an experimental system in Saccharomyces cerevisiae that enables selection of large-scale contractions of (CCTG)100 within the intron of a reporter gene and subsequent genetic analysis. Contractions exceeded 80 repeat units, causing the final repetitive tract to be well below the threshold for disease. We found that Rad51 and Rad52 are required for these massive contractions, indicating a mechanism that involves homologous recombination. Srs2 helicase was shown previously to stabilize CTG, CAG, and CGG repeats. Loss of Srs2 did not significantly affect CCTG contraction rates in unperturbed conditions. In contrast, loss of the RecQ helicase Sgs1 resulted in a 6-fold decrease in contraction rate with specific evidence that helicase activity is required for large-scale contractions. Using a genetic assay to evaluate chromosome arm loss, we determined that CCTG and reverse complementary CAGG repeats elevate the rate of chromosomal fragility compared to a low-repeat control. Overall, our results demonstrate that the genetic control of CCTG repeat contractions is notably distinct among disease-causing microsatellite repeat sequences.

OprF impacts Pseudomonas aeruginosa biofilm matrix eDNA levels in a nutrient-dependent manner.

The biofilm matrix is composed of exopolysaccharides, eDNA, membrane vesicles, and proteins. While proteomic analyses have identified numerous matrix proteins, their functions in the biofilm remain understudied compared to the other biofilm components. In the Pseudomonas aeruginosa biofilm, several studies have identified OprF as an abundant matrix protein and, more specifically, as a component of biofilm membrane vesicles. OprF is a major outer membrane porin of P. aeruginosa cells. However, current data describing the effects of OprF in the P. aeruginosa biofilm is limited. Here we identify a nutrient-dependent effect of OprF in static biofilms, whereby Δ oprF cells form significantly less biofilm than wild type when grown in media containing glucose or low sodium chloride concentrations. Interestingly, this biofilm defect occurs during late static biofilm formation and is not dependent on the production of PQS, which is responsible for outer membrane vesicle production. Furthermore, while biofilms lacking OprF contain approximately 60% less total biomass than those of wild type, the number of cells in these two biofilms is equivalent. We demonstrate that P. aeruginosa Δ oprF biofilms with reduced biofilm biomass contain less eDNA than wild-type biofilms. These results suggest that the nutrient-dependent effect of OprF is involved in the maintenance of mature P. aeruginosa biofilms by retaining eDNA in the matrix. IMPORTANCE: Many pathogens form biofilms, which are bacterial communities encased in an extracellular matrix that protects them against antibacterial treatments. The roles of several matrix components of the opportunistic pathogen Pseudomonas aeruginosa have been characterized. However, the effects of P. aeruginosa matrix proteins remain understudied and are untapped potential targets for antibiofilm treatments. Here we describe a conditional effect of the abundant matrix protein OprF on late-stage P. aeruginosa biofilms. A Δ oprF strain formed significantly less biofilm in low sodium chloride or with glucose. Interestingly, the defective Δ oprF biofilms did not exhibit fewer resident cells but contained significantly less extracellular DNA (eDNA) than wild type. These results suggest that OprF is involved in matrix eDNA retention in mature biofilms.

Advocating for a New Residency Application Process: A Student Perspective.

OBJECTIVE: The COVID-19 pandemic provided an opportunity for surgical residency programs to rethink their methods of evaluating and recruiting candidates. However, the past year has not been seamless, with a soaring number of applications, reports of programs and applicants having difficulty evaluating each other, and an increasingly uneven distribution of interviews among applicants. Consequently, many have called for national changes to the residency application process to address these longstanding concerns. RESULTS: Here, we review the evolving literature and advocate for the permanent adoption of visiting rotations, virtual interviews with a universal release date and data-driven attendance limits, and opportunities for in-person applicant visits. CONCLUSIONS: We believe these changes leverage the strengths of each format, allow for satisfactory bidirectional evaluation, and promote principles of justice, equity, diversity, and inclusion.

Screening for Primary Aldosteronism is Underutilized in Patients with Obstructive Sleep Apnea.

BACKGROUND: Resistant hypertension is common in patients with primary aldosteronism and in those with obstructive sleep apnea. Primary aldosteronism treatment improves sleep apnea. Despite Endocrine Society guidelines' inclusion of sleep apnea and hypertension co-diagnosis as a primary aldosteronism screening indication, the state of screening implementation is unknown. METHODS: All hypertensive adult patients with obstructive sleep apnea (n = 4751) at one institution between 2012 and 2020 were compared with a control cohort without sleep apnea (n = 117,815). We compared the association of primary aldosteronism diagnoses, risk factors, and screening between both groups. Patients were considered to have screening if they had a primary aldosteronism diagnosis or serum aldosterone or plasma renin activity evaluation. RESULTS: Obstructive sleep apnea patients were predominantly men and had higher body mass index. On multivariable analysis, hypertensive sleep apnea patients had higher odds of drug-resistant hypertension (odds ratio [OR] 2.70; P < .001) and hypokalemia (OR 1.26; P < .001) independent of body mass index, sex, and number of antihypertensive medications. Overall, sleep apnea patients were more likely to be screened for primary aldosteronism (OR 1.45; P < .001); however, few patients underwent screening whether they had sleep apnea or not (pre-guideline publication 7.8% vs 4.6%; post-guidelines 3.6% vs 4.6%; P < .01). Screening among eligible sleep apnea patients remained low prior to and after guideline publication (4.4% vs 3.4%). CONCLUSIONS: Obstructive sleep apnea is associated with primary aldosteronism risk factors without formal diagnosis, suggesting screening underutilization and underdiagnosis. Strategies are needed to increase screening adherence, as patients may benefit from treatment of concomitant primary aldosteronism to reduce sleep apnea severity and its associated cardiopulmonary morbidity.

Third year medical student knowledge gaps after a virtual surgical rotation.

INTRODUCTION: This study describes perceived knowledge gaps of third-year medical students after participating in a virtual surgical didactic rotation (EMLR) and shortened in-person surgery rotation during the COVID-19 Pandemic. METHODS: Open-ended and Likert questions were administered at the end of the virtual rotation and inperson-surgical rotation to medical students. Three blinded coders identified themes by semantic analysis. RESULTS: 82 students (51% of all MS3s) participated in the EMLR. Semantic analysis revealed gaps in perioperative management (Post-EMLR:18.4%, Post-Inpatient:26.5%), anatomy (Post-EMLR:8.2%, PostInpatient:26.5%). and surgical skills (Post-EMLR: 43.0%, Post-Inpatient: 44.1%). Students also described gaps related to OR etiquette (Post-EMLR: 12.2%, Post-Inpatient: 8.8%) and team dynamics/the hidden curriculum (Post- Inpatient:26.6%). There was a significant improvement in perceived confidence to perform inpatient tasks after completing the inpatient clinical experience (p ≤ 0.01). CONCLUSION: Virtual interactive didactics for cognitive skills development cannot replace a full clinical surgical experience for third-year medical students. Future curricula should address perceived gaps.