Demographics, diagnostics, treatment, and outcomes of patients presenting with acute groin hernia: 15-year multicentre retrospective cohort study.

BACKGROUND: Groin hernias commonly present acutely in high-risk populations and can be challenging to manage. This retrospective, observational study aimed to report on patient demographics and outcomes, following acute admissions with a groin hernia, in relation to contemporary investigative and management practices. METHODS: Adult (≥18 years old) patients who presented acutely with a groin hernia to nine National Health Service trusts in the north of England between 2002 and 2016 were included. Data were collected regarding patient demographics, radiological investigations, and operative intervention. The primary outcome of interest was 30-day inpatient mortality rate. RESULTS: Overall, 6165 patients with acute groin hernia were included (4698 inguinal and 1467 femoral hernias). There was a male preponderance (72.5 per cent) with median age of 73 years (interquartile range (i.q.r.) 58-82). The burden of patient co-morbidity increased over the study period (P < 0.001). Operative repair was performed in 2258 (55.1 per cent) of patients with an inguinal and 1321 (90.1 per cent) of patients with a femoral hernia. Bowel resection was more commonly required for femoral hernias (14.7 per cent) than inguinal hernias (3.5 per cent, P < 0.001) and in obstructed (14.6 versus 0.2 per cent, P < 0.001) or strangulated (58.4 versus 4.5 per cent, P < 0.001) hernias. The 30-day mortality rate was 3.1 per cent for the overall cohort and 3.9 per cent for those who underwent surgery. Bowel resection was associated with increased duration of hospital stay (P < 0.001) and 30-day inpatient mortality rate (P < 0.001). Following adjustment for confounding variables, advanced age, co-morbidity, obstruction, and strangulation were all associated with an increased 30-day mortality rate (all P < 0.001). CONCLUSION: Emergency hernia repair has high mortality rates. Advanced age and co-morbidity increase both duration of hospital stay and 30-day mortality rate.

A Graduate Medical Orientation Intervention Focused on Local Health Inequities.

Background: Residents must understand the social drivers of health in the communities they serve to deliver quality care. While resident orientation provides an opportunity to introduce residents to social and structural drivers of health, inequity, and care delivery relevant to the patient population in their new communities, many graduate medical education orientation curricula do not include this content. Objective: To report the development and implementation of a novel, patient-centered health equity orientation curriculum, including initial feasibility and acceptability data as well as preliminary self-reported outcomes. Methods: The curriculum was developed by academic faculty in collaboration with institutional and local health equity champions. Content centered on the history of inequities and racism within the local communities and included didactic presentations, asynchronous video, and virtual site visits to community resource groups. The curriculum was administered to all 2021 incoming Vanderbilt University Medical Center medical and surgical residents (N=270) over 2 half-days, both in-person and via Zoom. Data were collected anonymously via pre- and post-surveys. Results: A total of 216 residents (80% response rate) provided pre-survey response data, but only 138 residents (51.1%) provided post-survey data, including self-reported demographics (eg, underrepresented in medicine status) and level of agreement with 10 competency-based statements coded as pertaining to knowledge, skills, behaviors, or attitudes (KSBAs). Primary outcomes included improvement in residents' KSBAs from pre- to post-survey. The greatest increases in percentages occurred with content that was specific to local history and population. Conclusions: In a class of incoming residents, this study demonstrated feasibility, acceptability, and pre-post curriculum improvement in self-reported KSBAs when addressing health equity issues.

Mechanisms to Repair Stalled Topoisomerase II-DNA Covalent Complexes.

DNA topoisomerases regulate the topological state of DNA, relaxing DNA supercoils and resolving catenanes and knots that result from biologic processes, such as transcription and replication. DNA topoisomerase II (TOP2) enzymes achieve this by binding DNA and introducing an enzyme-bridged DNA double-strand break (DSB) where each protomer of the dimeric enzyme is covalently attached to the 5' end of the cleaved DNA via an active site tyrosine phosphodiester linkage. The enzyme then passes a second DNA duplex through the DNA break, before religation and release of the enzyme. However, this activity is potentially hazardous to the cell, as failure to complete religation leads to persistent TOP2 protein-DNA covalent complexes, which are cytotoxic. Indeed, this property of topoisomerase has been exploited in cancer therapy in the form of topoisomerase poisons which block the religation stage of the reaction cycle, leading to an accumulation of topoisomerase-DNA adducts. A number of parallel cellular processes have been identified that lead to removal of these covalent TOP2-DNA complexes, facilitating repair of the resulting protein-free DSB by standard DNA repair pathways. These pathways presumably arose to repair spontaneous stalled or poisoned TOP2-DNA complexes, but understanding their mechanisms also has implications for cancer therapy, particularly resistance to anti-cancer TOP2 poisons and the genotoxic side effects of these drugs. Here, we review recent progress in the understanding of the processing of TOP2 DNA covalent complexes, the basic components and mechanisms, as well as the additional layer of complexity posed by the post-translational modifications that modulate these pathways. SIGNIFICANCE STATEMENT: Multiple pathways have been reported for removal and repair of TOP2-DNA covalent complexes to ensure the timely and efficient repair of TOP2-DNA covalent adducts to protect the genome. Post-translational modifications, such as ubiquitination and SUMOylation, are involved in the regulation of TOP2-DNA complex repair. Small molecule inhibitors of these post-translational modifications may help to improve outcomes of TOP2 poison chemotherapy, for example by increasing TOP2 poison cytotoxicity and reducing genotoxicity, but this remains to be determined.

A Role for VCP/p97 in the Processing of Drug-Stabilized TOP2-DNA Covalent Complexes.

DNA topoisomerase II (TOP2) poisons induce protein-DNA crosslinks termed TOP2-DNA covalent complexes, in which TOP2 remains covalently bound to each end of an enzyme-induced double-strand DNA break (DSB) via a 5'-phosphotyrosyl bond. Repair of the enzyme-induced DSB first requires the removal of the TOP2 protein adduct, which, among other mechanisms, can be accomplished through the proteasomal degradation of TOP2. VCP/p97 is a AAA ATPase that utilizes energy from ATP hydrolysis to unfold protein substrates, which can facilitate proteasomal degradation by extracting target proteins from certain cellular structures (such as chromatin) and/or by aiding their translocation into the proteolytic core of the proteasome. In this study, we show that inhibition of VCP/p97 leads to the prolonged accumulation of etoposide-induced TOP2A and TOP2B complexes in a manner that is epistatic with the proteasomal pathway. VCP/p97 inhibition also reduces the etoposide-induced phosphorylation of histone H2A.X, indicative of fewer DSBs. This suggests that VCP/p97 is required for the proteasomal degradation of TOP2-DNA covalent complexes and is thus likely to be an important mediator of DSB repair after treatment with a TOP2 poison. SIGNIFICANCE STATEMENT: TOP2 poisons are chemotherapeutic agents used in the treatment of a range of cancers. A better understanding of how TOP2 poison-induced DNA damage is repaired could improve therapy with TOP2 poisons by increasing TOP2 poison cytotoxicity and reducing genotoxicity. The results presented herein suggest that repair of TOP2-DNA covalent complexes involves the protein segregase VCP/p97.

Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes.

DNA topoisomerase II (TOP2) is required for the unwinding and decatenation of DNA through the induction of an enzyme-linked double-strand break (DSB) in one DNA molecule and passage of another intact DNA duplex through the break. Anticancer drugs targeting TOP2 (TOP2 poisons) prevent religation of the DSB and stabilize a normally transient intermediate of the TOP2 reaction mechanism called the TOP2-DNA covalent complex. Subsequently, TOP2 remains covalently bound to each end of the enzyme-bridged DSB, which cannot be repaired until TOP2 is removed from the DNA. One removal mechanism involves the proteasomal degradation of the TOP2 protein, leading to the liberation of a protein-free DSB. Proteasomal degradation is often regulated by protein ubiquitination, and here we show that inhibition of ubiquitin-activating enzymes reduces the processing of TOP2A- and TOP2B-DNA complexes. Depletion or inhibition of ubiquitin-activating enzymes indicated that ubiquitination was required for the liberation of etoposide-induced protein-free DSBs and is therefore an important layer of regulation in the repair of TOP2 poison-induced DNA damage. TOP2-DNA complexes stabilized by etoposide were shown to be conjugated to ubiquitin, and this was reduced by inhibition or depletion of ubiquitin-activating enzymes. SIGNIFICANCE STATEMENT: There is currently great clinical interest in the ubiquitin-proteasome system and ongoing development of specific inhibitors. The results in this paper show that the therapeutic cytotoxicity of DNA topoisomerase II (TOP2) poisons can be enhanced through combination therapy with ubiquitin-activating enzyme inhibitors or by specific inhibition of the BMI/RING1A ubiquitin ligase, which would lead to increased cellular accumulation or persistence of TOP2-DNA complexes.

The Deubiquitinating Enzyme Inhibitor PR-619 is a Potent DNA Topoisomerase II Poison.

2,6-Diaminopyridine-3,5-bis(thiocyanate) (PR-619) is a broad-spectrum deubiquitinating enzyme (DUB) inhibitor that has been employed in cell-based studies as a tool to investigate the role of ubiquitination in various cellular processes. Here, we demonstrate that in addition to its action as a DUB inhibitor, PR-619 is a potent DNA topoisomerase II (TOP2) poison, inducing both DNA topoisomerase IIα (TOP2A) and DNA topoisomerase IIß (TOP2B) covalent DNA complexes with similar efficiency to the archetypal TOP2 poison etoposide. However, in contrast to etoposide, which induces TOP2-DNA complexes with a pan-nuclear distribution, PR-619 treatment results in nucleolar concentration of TOP2A and TOP2B. Notably, neither the induction of TOP2-DNA covalent complexes nor their nucleolar concentration are due to TOP2 hyperubiquitination since both occur even under conditions of depleted ubiquitin. Like etoposide, since PR-619 affected TOP2 enzyme activity in in vitro enzyme assays as well as in live cells, we conclude that PR-619 interacts directly with TOP2A and TOP2B. The concentration at which PR-619 exhibits robust cellular DUB inhibitor activity (5-20 µM) is similar to the lowest concentration at which TOP2 poison activity was detected (above 20 µM), which suggests that caution should be exercised when employing this DUB inhibitor in cell-based studies.

Intercalating TOP2 Poisons Attenuate Topoisomerase Action at Higher Concentrations.

Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase IIß (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.

Development of an interprofessional clinical learning environment report card.

Multiple factors in the learning environment can encourage or impede student learning. Unanswered questions regarding the shared learning environment for graduate nursing and medical education and the desire for an ongoing improvement process drove creation of an interprofessional collaborative and development of an Interprofessional Clinical Learning Environment Report Card (I-CLERC) at one U.S. academic medical center. The I-CLERC offers a process and a product for institutionalizing a shared assessment tool to inform improvement efforts, track progress and promote accountability. In addition, it enhances interprofessional collaboration, with students and faculty from both nursing and medicine working together to define excellence, monitor performance, and identify areas for improvement in the shared clinical learning environment. The purpose of this manuscript is to describe development and implementation of an interdisciplinary, institutional collaborative for ongoing evaluation of the shared clinical learning environment.

TOP2B: The First Thirty Years.

Type II DNA topoisomerases (EC 5.99.1.3) are enzymes that catalyse topological changes in DNA in an ATP dependent manner. Strand passage reactions involve passing one double stranded DNA duplex (transported helix) through a transient enzyme-bridged break in another (gated helix). This activity is required for a range of cellular processes including transcription. Vertebrates have two isoforms: topoisomerase IIα and ß. Topoisomerase IIß was first reported in 1987. Here we review the research on DNA topoisomerase IIß over the 30 years since its discovery.

Effect of TDP2 on the Level of TOP2-DNA Complexes and SUMOylated TOP2-DNA Complexes.

DNA topoisomerase II (TOP2) activity involves a normally transient double-strand break intermediate in which the enzyme is coupled to DNA via a 5'-phosphotyrosyl bond. However, etoposide and other topoisomerase drugs poison the enzyme by stabilising this enzyme-bridged break, resulting in the accumulation of TOP2-DNA covalent complexes with cytotoxic consequences. The phosphotyrosyl diesterase TDP2 appears to be required for efficient repair of this unusual type of DNA damage and can remove 5'-tyrosine adducts from a double-stranded oligonucleotide substrate. Here, we adapt the trapped in agarose DNA immunostaining (TARDIS) assay to investigate the role of TDP2 in the removal of TOP2-DNA complexes in vitro and in cells. We report that TDP2 alone does not remove TOP2-DNA complexes from genomic DNA in vitro and that depletion of TDP2 in cells does not slow the removal of TOP2-DNA complexes. Thus, if TDP2 is involved in repairing TOP2 adducts, there must be one or more prior steps in which the protein-DNA complex is processed before TDP2 removes the remaining 5' tyrosine DNA adducts. While this is partly achieved through the degradation of TOP2 adducts by the proteasome, a proteasome-independent mechanism has also been described involving the SUMOylation of TOP2 by the ZATT E3 SUMO ligase. The TARDIS assay was also adapted to measure the effect of TDP2 knockdown on levels of SUMOylated TOP2-DNA complexes, which together with levels of double strand breaks were unaffected in K562 cells following etoposide exposure and proteasomal inhibition.

The differential expression of omega-3 and omega-6 fatty acid metabolising enzymes in colorectal cancer and its prognostic significance.

BACKGROUND: Colorectal cancer is a common malignancy and one of the leading causes of cancer-related deaths. The metabolism of omega fatty acids has been implicated in tumour growth and metastasis. METHODS: This study has characterised the expression of omega fatty acid metabolising enzymes CYP4A11, CYP4F11, CYP4V2 and CYP4Z1 using monoclonal antibodies we have developed. Immunohistochemistry was performed on a tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosa. RESULTS: The differential expression of CYP4A11 and CYP4F11 showed a strong association with survival in both the whole patient cohort (hazard ratio (HR)=1.203, 95% CI=1.092-1.324, χ2=14.968, P=0.001) and in mismatch repair-proficient tumours (HR=1.276, 95% CI=1.095-1.488, χ2=9.988, P=0.007). Multivariate analysis revealed that the differential expression of CYP4A11 and CYP4F11 was independently prognostic in both the whole patient cohort (P=0.019) and in mismatch repair proficient tumours (P=0.046). CONCLUSIONS: A significant and independent association has been identified between overall survival and the differential expression of CYP4A11 and CYP4F11 in the whole patient cohort and in mismatch repair-proficient tumours.

Characterisation of the oxysterol metabolising enzyme pathway in mismatch repair proficient and deficient colorectal cancer.

Oxysterols are oxidised derivatives of cholesterol, formed by the enzymatic activity of several cytochrome P450 enzymes and tumour-derived oxysterols have been implicated in tumour growth and survival. The aim of this study was to profile the expression of oxysterol metabolising enzymes in primary colorectal cancer and assess the association between expression and prognosis.Immunohistochemistry was performed on a colorectal cancer tissue microarray containing 650 primary colorectal cancers using monoclonal antibodies to CYP2R1, CYP7B1, CYP8B1, CYP27A1, CYP39A1, CYP46A1 and CYP51A1, which we have developed. Unsupervised hierarchical cluster analysis was used to examine the overall relationship of oxysterol metabolising enzyme expression with outcome and based on this identify an oxysterol metabolising enzyme signature associated with prognosis.Cluster analysis of the whole patient cohort identified a good prognosis group (mean survival=146 months 95% CI 127-165 months) that had a significantly better survival (χ2=12.984, p<0.001, HR=1.983, 95% CI 1.341-2.799) than the poor prognosis group (mean survival=107 months, 95% CI 98-123 months). For the mismatch repair proficient cohort, the good prognosis group had a significantly better survival (χ2=8.985, p=0.003, HR=1.845, 95% CI 1.227-2.774) than the poor prognosis group. Multi-variate analysis showed that cluster group was independently prognostically significant in both the whole patient cohort (p=0.02, HR=1.554, 95% CI 1.072-2.252) and the mismatch repair proficient group (p=0.04, HR=1.530, 95% CI 1.014-2.310).Individual oxysterol metabolising enzymes are overexpressed in colorectal cancer and an oxysterol metabolising enzyme expression profile associated with prognosis has been identified in the whole patient cohort and in mismatch repair proficient colorectal cancers.

Formal training in vaccine safety to address parental concerns not routinely conducted in U.S. pediatric residency programs.

OBJECTIVE: To determine if U.S. pediatric residency programs provide formal training in vaccine safety to address parental vaccine concerns. METHODS: An electronic survey was mailed to all members of the Association of Pediatric Program Directors (APPD) to assess (1) if U.S. pediatric residency programs were providing formal vaccine safety training, (2) the content and format of the training if provided, and (3) interest in a training module for programs without training. Two follow-up surveys were mailed at 2 week intervals. Responses to the survey were collected at 4 weeks following the last mailing and analyzed. Logistic regression was used to assess the impact of program size on the likelihood of vaccine safety training. Pearson's chi square was used to compare programs with and without formal vaccine safety training in 5 U.S. regions. RESULTS: The survey was sent to 199 APPD members; 92 completed the survey (response rate 46.2%). Thirty-eight respondents (41%) had formal training in vaccine safety for pediatric residents at their programs; 54 (59%) did not. Of those that did not, the majority (81.5%) were interested in formal vaccine safety training for their residents. Of all respondents, 78% agreed that training in vaccine safety was a high priority for resident education. Thirty-five percent of all respondents agreed that local parental attitudes about vaccines influenced the likelihood of formal vaccine safety training. CONCLUSION: Most pediatric residency programs surveyed do not include formal training on vaccine safety; yet, such training is supported by pediatric residency program directors as a priority for pediatric residents.

Pediatric palliative care instruction for residents: an introduction to IPPC.

A 1-day training event for pediatric residents with interdisciplinary staff was held, which was modeled after the Initiative for Pediatric Palliative Care (IPPC). Training included relational communication, cultural humility, pain-symptom management, family-centered care, team problem solving, and strategic planning using didactic, small group, and plenary platforms. Two bereaved parents were co-learners and trainers. Twenty-six interdisciplinary staff participated. A positive impact was measured in new knowledge gained, value in collaborative learning with health care professionals and families, and ability to work with professionals outside participants' own unit. Confidence to advocate for improved pediatric palliative care was also noted. The IPPC curriculum is easily adapted for resident education. Incorporating family members as co-learners and teachers is valuable. Advocacy for pediatric palliative care may follow this type of experience.

The effect of protease inhibition on the temporal stability of NT-proBNP in feline plasma at room temperature.

OBJECTIVES: To evaluate whether the addition of a protease inhibitor (PI) to feline plasma improves the temporal stability of N-terminal pro-B-type natriuretic peptide (NT-proBNP). ANIMALS, MATERIALS AND METHODS: Forty-four EDTA blood samples were collected from 42 cats with cardiac disease or hyperthyroidism. Samples were separated within 15 min of collection and the plasma stored at -80 °C. Samples were thawed and each separated into 2 aliquots, one of which was mixed with PI. Each was subdivided into 5 aliquots, which remained at room temperature (RT) for zero, 24, 48, 96 or 120 h before being returned to storage at -80 °C. NT-proBNP was measured using a commercially-available ELISA. RESULTS: There was no difference in NT-proBNP measurements between plain and PI samples at time zero (P = 0.836) or 24 h (P = 0.293). At subsequent time-points NT-proBNP was higher in the PI than the plain samples (P < 0.05). An overall effect of time at RT was detected for plain (P < 0.001) and PI samples (P < 0.001). CONCLUSIONS: The addition of PI to feline plasma significantly reduces, but does not eliminate, degradation of NT-proBNP at RT. This degradation occurs over a time course comparable to postal transportation of samples for laboratory analysis and may alter the clinical interpretation of results.

A simplified observation tool for residents in the outpatient clinic.

BACKGROUND: The Accreditation Council for Graduate Medical Education promotes direct observation of residents as a key assessment tool for competency in patient care, professionalism, and communication skills. Although tools exist, validity and reliability have not been demonstrated for most, and many tools may have limited feasibility because of time constraints and other reasons. We conducted a study to measure feasibility of a simplified observation tool to evaluate these competencies and provide timely feedback. METHODS: In the pediatric resident continuity clinic of a large children's hospital, we used a direct observation form with a 3-point scale for 16 items in the domains of patient care, professionalism, and communication skills. The form was divided by portion of visit, with specific items mapped to 1 or more of the competencies, and was used to provide direct oral feedback to the resident. Faculty and residents completed surveys rating the process (ease of use, satisfaction, and self-assessed usefulness) on a 5-point Likert scale. RESULTS: The study encompassed 89 surveys completed by attending physicians; 98% (87 of 89) of the time the form was easy to use, 99% (88) of the time its use did not interfere with patient flow, and 93% (83) of the observations provided useful information for resident feedback. Residents completed 70 surveys, with the majority (69%, 48) reporting they were comfortable about being observed by an attending physician; 87% (61) thought that direct observation did not significantly affect their efficiency. Ninety-seven percent of the time (68) residents reported that direct observation provided useful feedback. CONCLUSION: The data suggest the form was well-received by both faculty and residents, and enabled attending physicians to provide useful feedback.