DNA methylation is of paramount importance for mammalian embryonic development. DNA methylation has numerous functions: it is implicated in the repression of transposons and genes, but is also ...associated with actively transcribed gene bodies and, in some cases, with gene activation per se. In recent years, sensitive technologies have been developed that allow the interrogation of DNA methylation patterns from a small number of cells. The use of these technologies has greatly improved our knowledge of DNA methylation dynamics and heterogeneity in embryos and in specific tissues. Combined with genetic analyses, it is increasingly apparent that regulation of DNA methylation erasure and (re-)establishment varies considerably between different developmental stages. In this Review, we discuss the mechanisms and functions of DNA methylation and demethylation in both mice and humans at CpG-rich promoters, gene bodies and transposable elements. We highlight the dynamic erasure and re-establishment of DNA methylation in embryonic, germline and somatic cell development. Finally, we provide insights into DNA methylation gained from studying genetic diseases.
This 2017 Presidential Address for the Association for Academic Surgery was delivered on February 8, 2017. It addresses the difficult topic of gender disparities in surgery. Mixing empirical data ...with personal anecdotes, Dr. Caprice Greenberg provides an insightful overview of this difficult challenge facing the surgical discipline and practical advice on how we can begin to address it.
The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Overexpression of either PAX-FOXO1s ...results in related cell transformation in animal models. Yet, in patients the two structural genetic aberrations they derived from are associated with distinct pathological manifestations. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 binding results in greater recruitment of the H3K27ac activation mark than PAX3-FOXO1 binding and is accompanied by greater transcriptional activation of neighbouring genes. These effects are associated with a PAX-FOXO1-specific alteration in the expression of genes regulating cell shape and the cell cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into S phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into M phase, and causes increased DNA damage. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the genomic occupancy and transcriptional activity of PAX3-FOXO1 and PAX7-FOXO1.
Postoperative complications remain common after surgery, but little is known about the extent of variation in operative technical skill and whether variation is associated with patient outcomes.
To ...examine the (1) variation in technical skill scores of practicing surgeons, (2) association between technical skills and patient outcomes, and (3) amount of variation in patient outcomes explained by a surgeon's technical skill.
In this quality improvement study, 17 practicing surgeons submitted a video of a laparoscopic right hemicolectomy that was then rated by at least 10 blinded peer surgeons and 2 expert raters. The association between surgeon technical skill scores and risk-adjusted outcomes was examined using data from the American College of Surgeons National Surgical Quality Improvement Program. The association between technical skill scores and outcomes was examined for colorectal procedures and noncolorectal procedures (ie, assessed on whether technical skills demonstrated during colectomy were associated with patient outcomes across other cases). In addition, the proportion of patient outcomes explained by technical skill scores was examined using robust regression techniques. The study was conducted from September 23, 2016, to February 10, 2018; data analysis was performed from November 2018 to January 2019.
Colorectal and noncolorectal procedures.
Any complication, mortality, unplanned hospital readmission, unplanned reoperation related to principal procedure, surgical site infection, and death or serious morbidity.
Of the 17 surgeons included in the study, 13 were men (76%). The participants had a range from 1 to 28 years in surgical practice (median, 11 years). Based on 10 or more reviewers per video and with a maximum quality score of 5, overall technical skill scores ranged from 2.8 to 4.6. From 2014 to 2016, study participants performed a total of 3063 procedures (1120 colectomies). Higher technical skill scores were significantly associated with lower rates of any complication (15.5% vs 20.6%, P = .03; Spearman rank-order correlation coefficient r = -0.54, P = .03), unplanned reoperation (4.7% vs 7.2%, P = .02; r = -0.60, P = .01), and a composite measure of death or serious morbidity (15.9% vs 21.4%, P = .02; r = -0.60, P = .01) following colectomy. Similar associations were found between colectomy technical skill scores and patient outcomes for all types of procedures performed by a surgeon. Overall, technical skill scores appeared to account for 25.8% of the variation in postcolectomy complication rates and 27.5% of the variation when including noncolectomy complication rates.
The findings of this study suggest that there is wide variation in technical skill among practicing surgeons, accounting for more than 25% of the variation in patient outcomes. Higher colectomy technical skill scores appear to be associated with lower complication rates for colectomy and for all other procedures performed by a surgeon. Efforts to improve surgeon technical skills may result in better patient outcomes.
Computer vision was used to predict expert performance ratings from surgeon hand motions for tying and suturing tasks.
Existing methods, including the objective structured assessment of technical ...skills (OSATS), have proven reliable, but do not readily discriminate at the task level. Computer vision may be used for evaluating distinct task performance throughout an operation.
Open surgeries was videoed and surgeon hands were tracked without using sensors or markers. An expert panel of 3 attending surgeons rated tying and suturing video clips on continuous scales from 0 to 10 along 3 task measures adapted from the broader OSATS: motion economy, fluidity of motion, and tissue handling. Empirical models were developed to predict the expert consensus ratings based on the hand kinematic data records.
The predicted versus panel ratings for suturing had slopes from 0.73 to 1, and intercepts from 0.36 to 1.54 (Average R2 = 0.81). Predicted versus panel ratings for tying had slopes from 0.39 to 0.88, and intercepts from 0.79 to 4.36 (Average R2 = 0.57). The mean square error among predicted and expert ratings was consistently less than the mean squared difference among individual expert ratings and the eventual consensus ratings.
The computer algorithm consistently predicted the panel ratings of individual tasks, and were more objective and reliable than individual assessment by surgical experts.
Objective Coronary artery bypass grafting (CABG) is the operation most commonly performed by cardiac surgeons. There are few contemporary data examining evolving patient characteristics and surgical ...outcomes of isolated CABG. We used the Society of Thoracic Surgeons adult cardiac surgery database to characterize trends in patient characteristics and outcomes after CABG over the past decade. Methods From 2000 to 2009, 1,497,254 patients underwent isolated primary CABG at Society of Thoracic Surgeons participating institutions. Demographics, operative characteristics, and postoperative outcomes were assessed, and risk-adjusted outcomes were calculated. Results Compared with the year 2000, patients undergoing isolated primary CABG in 2009 were more likely to have diabetes mellitus (33% vs 40%) and hypertension (71% vs 85%). There were clinically insignificant differences in age, gender, and body surface area. Between 2000 and 2009, there has been a 6.3% and 19.5% increase in the preoperative use of aspirin and beta-blockers, respectively. Between 2004 and 2009, there was a 7.8% increase in the use of angiotension-converting enzyme inhibitors preoperatively. Furthermore, between 2005 and 2009 there was a 3.8% increase in the use of statins preoperatively. The median number of distal anastomoses performed was unchanged between 2000 and 2009 (3; interquartile range, 2–4). There was a significant increase in the use of the internal thoracic artery (88% in 2000 vs 95% in 2009). The predicted mortality rates of 2.3% were consistent between 2000 and 2009. The observed mortality rate over this period declined from 2.4% in 2000 to 1.9% in 2009 representing a relative risk reduction of 24.4%. The incidence of postoperative stroke decreased significantly from 1.6% to 1.2%, representing a risk reduction of 26.4%. There was also a 9.2% relative reduction in the risk of reoperation for bleeding and a 32.9% relative risk reduction in the incidence of sternal wound infection. Conclusions Over the past decade, the risk profile of patients undergoing CABG has changed, with fewer smokers, more diabetic patients, and better medical therapy characterizing patients referred for surgical coronary revascularization. The left internal thoracic artery is nearly universally used and outcomes have improved substantially, with a significant decline in postoperative mortality and morbidity.
DNA methylation is an epigenetic mark that is associated with transcriptional repression of transposable elements and protein-coding genes. Conversely, transcriptionally active regulatory regions are ...strongly correlated with histone 3 lysine 4 di- and trimethylation (H3K4m2/m3). We previously showed that Arabidopsis thaliana plants with mutations in the H3K4m2/m3 demethylase JUMONJI 14 (JMJ14) exhibit a mild reduction in RNA-directed DNA methylation (RdDM) that is associated with an increase in H3K4m2/m3 levels. To determine whether this incomplete RdDM reduction was the result of redundancy with other demethylases, we examined the genetic interaction of JMJ14 with another class of H3K4 demethylases: lysine-specific demethylase 1-like 1 and lysine-specific demethylase 1-like 2 (LDL1 and LDL2). Genome-wide DNA methylation analyses reveal that both families cooperate to maintain RdDM patterns. ChIP-seq experiments show that regions that exhibit an observable DNA methylation decrease are co-incidental with increases in H3K4m2/m3. Interestingly, the impact on DNA methylation was stronger at DNA-methylated regions adjacent to H3K4m2/m3-marked protein-coding genes, suggesting that the activity of H3K4 demethylases may be particularly crucial to prevent spreading of active epigenetic marks. Finally, RNA sequencing analyses indicate that at RdDM targets, the increase of H3K4m2/m3 is not generally associated with transcriptional de-repression. This suggests that the histone mark itself--not transcription--impacts the extent of RdDM.
The aim of the study was to (1) assess differences in how male and female general surgery residents utilize duty-hour regulations and experience aspects of burnout and psychological well-being, and ...(2) to explore reasons why these differing experiences exist.
There may be differences in how women and men enter, experience, and leave residency programs.
A total of 7395 residents completed a survey (response rate = 99%). Logistic regression models were developed to examine the association between gender and resident outcomes. Semistructured interviews were conducted with 42 faculty and 56 residents. Transcripts were analyzed thematically using a constant comparative approach.
Female residents reported more frequently staying in the hospital >28 hours or working >80 hours in a week (≥3 times in a month, P < 0.001) and more frequently feeling fatigued and burned out from their work (P < 0.001), but less frequently "treating patients as impersonal objects" or "not caring what happens" to them (P < 0.001). Women reported more often having experienced many aspects of poor psychological well-being such as feeling unhappy and depressed or thinking of themselves as worthless (P < 0.01). In adjusted analyses, associations remained significant. Themes identified in the qualitative analysis as possible contributory factors to gender differences include a lack of female mentorship/leadership, dual-role responsibilities, gender blindness, and differing pressures and approaches to patient care.
Female residents report working more, experiencing certain aspects of burnout more frequently, and having poorer psychological well-being. Qualitative themes provide insights into possible cultural and programmatic shifts to address the concerns for female residents.
Prognostic stratification is the cornerstone of management in nonmetastatic prostate cancer (PCa). However, existing prognostic models are inadequate-often using treatment outcomes rather than ...survival, stratifying by broad heterogeneous groups and using heavily treated cohorts. To address this unmet need, we developed an individualised prognostic model that contextualises PCa-specific mortality (PCSM) against other cause mortality, and estimates the impact of treatment on survival.
Using records from the United Kingdom National Cancer Registration and Analysis Service (NCRAS), data were collated for 10,089 men diagnosed with nonmetastatic PCa between 2000 and 2010 in Eastern England. Median follow-up was 9.8 years with 3,829 deaths (1,202 PCa specific). Totals of 19.8%, 14.1%, 34.6%, and 31.5% of men underwent conservative management, prostatectomy, radiotherapy (RT), and androgen deprivation monotherapy, respectively. A total of 2,546 men diagnosed in Singapore over a similar time period represented an external validation cohort. Data were randomly split 70:30 into model development and validation cohorts. Fifteen-year PCSM and non-PCa mortality (NPCM) were explored using separate multivariable Cox models within a competing risks framework. Fractional polynomials (FPs) were utilised to fit continuous variables and baseline hazards. Model accuracy was assessed by discrimination and calibration using the Harrell C-index and chi-squared goodness of fit, respectively, within both validation cohorts. A multivariable model estimating individualised 10- and 15-year survival outcomes was constructed combining age, prostate-specific antigen (PSA), histological grade, biopsy core involvement, stage, and primary treatment, which were each independent prognostic factors for PCSM, and age and comorbidity, which were prognostic for NPCM. The model demonstrated good discrimination, with a C-index of 0.84 (95% CI: 0.82-0.86) and 0.84 (95% CI: 0.80-0.87) for 15-year PCSM in the UK and Singapore validation cohorts, respectively, comparing favourably to international risk-stratification criteria. Discrimination was maintained for overall mortality, with C-index 0.77 (95% CI: 0.75-0.78) and 0.76 (95% CI: 0.73-0.78). The model was well calibrated with no significant difference between predicted and observed PCa-specific (p = 0.19) or overall deaths (p = 0.43) in the UK cohort. Key study limitations were a relatively small external validation cohort, an inability to account for delayed changes to treatment beyond 12 months, and an absence of tumour-stage subclassifications.
'PREDICT Prostate' is an individualised multivariable PCa prognostic model built from baseline diagnostic information and the first to our knowledge that models potential treatment benefits on overall survival. Prognostic power is high despite using only routinely collected clinicopathological information.
Genomic imprinting affects gene expression in a parent-of-origin manner and has a profound impact on complex traits including growth and behavior. While the rat is widely used to model human ...pathophysiology, few imprinted genes have been identified in this murid. To systematically identify imprinted genes and genomic imprints in the rat, we use low input methods for genome-wide analyses of gene expression and DNA methylation to profile embryonic and extraembryonic tissues at allele-specific resolution.
We identify 14 and 26 imprinted genes in these tissues, respectively, with 10 of these genes imprinted in both tissues. Comparative analyses with mouse reveal that orthologous imprinted gene expression and associated canonical DNA methylation imprints are conserved in the embryo proper of the Muridae family. However, only 3 paternally expressed imprinted genes are conserved in the extraembryonic tissue of murids, all of which are associated with non-canonical H3K27me3 imprints. The discovery of 8 novel non-canonical imprinted genes unique to the rat is consistent with more rapid evolution of extraembryonic imprinting. Meta-analysis of novel imprinted genes reveals multiple mechanisms by which species-specific imprinted expression may be established, including H3K27me3 deposition in the oocyte, the appearance of ZFP57 binding motifs, and the insertion of endogenous retroviral promoters.
In summary, we provide an expanded list of imprinted loci in the rat, reveal the extent of conservation of imprinted gene expression, and identify potential mechanisms responsible for the evolution of species-specific imprinting.
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