Genome-wide association studies and Mendelian randomization analyses provide insights into the causes of early-onset colorectal cancer.

BACKGROUND: The incidence of early-onset colorectal cancer (EOCRC; diagnosed <50 years of age) is rising globally; however, the causes underlying this trend are largely unknown. CRC has strong genetic and environmental determinants, yet common genetic variants and causal modifiable risk factors underlying EOCRC are unknown. We conducted the first EOCRC-specific genome-wide association study (GWAS) and Mendelian randomization (MR) analyses to explore germline genetic and causal modifiable risk factors associated with EOCRC. PATIENTS AND METHODS: We conducted a GWAS meta-analysis of 6176 EOCRC cases and 65 829 controls from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), the Colorectal Transdisciplinary Study (CORECT), the Colon Cancer Family Registry (CCFR), and the UK Biobank. We then used the EOCRC GWAS to investigate 28 modifiable risk factors using two-sample MR. RESULTS: We found two novel risk loci for EOCRC at 1p34.1 and 4p15.33, which were not previously associated with CRC risk. We identified a deleterious coding variant (rs36053993, G396D) at polyposis-associated DNA repair gene MUTYH (odds ratio 1.80, 95% confidence interval 1.47-2.22) but show that most of the common genetic susceptibility was from noncoding signals enriched in epigenetic markers present in gastrointestinal tract cells. We identified new EOCRC-susceptibility genes, and in addition to pathways such as transforming growth factor (TGF) ß, suppressor of Mothers Against Decapentaplegic (SMAD), bone morphogenetic protein (BMP) and phosphatidylinositol kinase (PI3K) signaling, our study highlights a role for insulin signaling and immune/infection-related pathways in EOCRC. In our MR analyses, we found novel evidence of probable causal associations for higher levels of body size and metabolic factors-such as body fat percentage, waist circumference, waist-to-hip ratio, basal metabolic rate, and fasting insulin-higher alcohol drinking, and lower education attainment with increased EOCRC risk. CONCLUSIONS: Our novel findings indicate inherited susceptibility to EOCRC and suggest modifiable lifestyle and metabolic targets that could also be used to risk-stratify individuals for personalized screening strategies or other interventions.

Third year medical students impersonalize and hedge when providing negative upward feedback to clinical faculty.

Medical students provide clinical teaching faculty with feedback on their skills as educators through anonymous surveys at the end of their clerkship rotation. Because faculty are in a position of power, students are hesitant to provide candid feedback. Our objective was to determine if medical students were willing to provide negative upward feedback to clinical faculty and describe how they conveyed their feedback. A qualitative analysis of third year medical students' open-ended comments from evaluations of six clerkships was performed using politeness theory as a conceptual framework. Students were asked to describe how the clerkship enhanced their learning and how it could be improved. Midway through the academic year, instructions to provide full names of faculty/residents was added. Overall, there were significantly more comments on what worked well than suggestions for improvement regarding faculty/residents. Instructing students to name-names increased the rate of naming from 35% to 75% for what worked well and from 13% to 39% for suggestions for improvement. Hedging language was included in 61% of suggestions for improvement, but only 2% of what worked well. Students described the variability of their experience, used passive language and qualified negative experiences with positive ones. Medical students may use linguistic strategies, such as impersonalizing and hedging, to mitigate the impact of negative upward feedback. Working towards a culture that supports upward feedback would allow students to feel more comfortable providing candid comments about their experience.

RAD51 and DMC1 form mixed complexes associated with mouse meiotic chromosome cores and synaptonemal complexes.

The eukaryotic RecA homologues RAD51 and DMC1 function in homology recognition and formation of joint-molecule recombination intermediates during yeast meiosis. The precise immunolocalization of these two proteins on the meiotic chromosomes of plants and animals has been complicated by their high degree of identity at the amino acid level. With antibodies that have been immunodepleted of cross-reactive epitopes, we demonstrate that RAD51 and DMC1 have identical distribution patterns in extracts of mouse spermatocytes in successive prophase I stages, suggesting coordinate functionality. Immunofluorescence and immunoelectron microscopy with these antibodies demonstrate colocalization of the two proteins on the meiotic chromosome cores at early prophase I. We also show that mouse RAD51 and DMC1 establish protein-protein interactions with each other and with the chromosome core component COR1(SCP3) in a two-hybrid system and in vitro binding analyses. These results suggest that the formation of a multiprotein recombination complex associated with the meiotic chromosome cores is essential for the development and fulfillment of the meiotic recombination process.

Characterisation of organisational issues in paediatric clinical ethics consultation: a qualitative study.

BACKGROUND: The traditional approach to resolving ethics concerns may not address underlying organisational issues involved in the evolution of these concerns. This represents a missed opportunity to improve quality of care "upstream". The purpose of this study was to understand better which organisational issues may contribute to ethics concerns. METHODS: Directed content analysis was used to review ethics consultation notes from an academic children's hospital from 1996 to 2006 (N = 71). The analysis utilised 18 categories of organisational issues derived and modified from published quality improvement protocols. RESULTS: Organisational issues were identified in 68 of the 71 (96%) ethics consult notes across a range of patient settings and reasons for consultation. Thirteen of the 18 categories of organisational issues were identified and there was a median of two organisational issues per consult note. The most frequently identified organisational issues were informal organisational culture (eg, collective practices and approaches to situations with ethical dimensions that are not guided by policy), policies and procedures (eg, staff knows policy and/or procedural guidelines for an ethical concern but do not follow it) and communication (eg, communication about critical information, orders, or hand-offs repeatedly does not occur among services). CONCLUSIONS: Organisational issues contribute to ethical concerns that result in clinical ethics consults. Identifying and addressing organisational issues such as informal culture and communication may help decrease the recurrence of future similar ethics concerns.

Incoming Interns Perceived Preparedness for Core Entrustable Professional Activities.

INTRODUCTION: The AAMC described 13 core entrustable professional activities (EPAs) for which every graduating medical student should perform proficiently on day 1 of residency. We studied how prepared starting interns felt in the core EPAs. METHODS: Interns from a diverse health system were surveyed on how well medical school prepared them in the 13 core EPAs. Data were collected on type of medical school, participation in an acting/sub-internship (AI/SI), knowledge of EPAs, and participation in an EPA experience. RESULTS: We collected 224 surveys out of 384 (58%) interns. 61.2% attended allopathic, 14.6% attended osteopathic, and 24.2% attended international schools. 67% had not heard of EPAs. 29% had an EPA experience of which 82% were required. 80% or more felt prepared in all EPAs except orders (60.7%) and handovers (73%). Allopathic interns were significantly more likely to have heard of EPAs and participated in an EPA experience than international. Allopathic interns felt more prepared than international in oral presentations and evidence-based medicine. Interns who participated in an EPA experience felt more prepared for oral presentation and evidence-based medicine. There were small but significant differences in feeling prepared in certain EPAs and types of AI/SI taken. CONCLUSION: The majority of interns entering residency have not heard of EPAs with fewer than 1/3 of interns participating in an EPA experience. International graduates were less likely to be aware or have experience with EPAs and report being less prepared in oral presentation and evidence-based medicine compared to allopathic graduates.

Incorporation of an Interprofessional Palliative Care-Ethics Experience Into a Required Critical Care Acting Internship.

Introduction: The literature documents inadequate palliative medicine training in undergraduate and graduate medical education. As the population lives longer, many people will experience multiple chronic illnesses and the associated symptom burden. All physicians involved in clinical care of patients need to be equipped with the knowledge, attitudes, and skills necessary to provide palliative care, yet most physicians do not feel adequately prepared. We designed a curriculum to provide a meaningful palliative care-ethics (PCE) clinical experience to prepare senior medical students for future practice regardless of specialty choice. Methods: The Zucker School of Medicine at Hofstra/Northwell integrated a PCE experience into the required 4-week acting internship in critical care (AICC). Students met weekly with an interprofessional faculty member and presented clinical cases focusing on communication and/or bioethical challenges. Faculty facilitators ensured that the presentations integrated discussion of communication skills. During the final session, students shared written reflections. Students were invited to complete a satisfaction survey postrotation and 1 year after graduation. Results: The curriculum was evaluated positively by the graduating classes of 2015 (n = 28) and 2016 (n = 56) at the end of the course and 1 year postgraduation. Qualitative analysis of the class of 2018 fourth-year students' reflective writing demonstrated themes of role modeling, suffering, family, and goals of care. Discussion: It is feasible to incorporate an interprofessional PCE experience into a required AICC. Students indicated a better understanding of palliative care and, at 1 year postgraduation, reported feeling comfortable caring for patients with serious illness.

Paramecium genome survey: a pilot project.

A consortium of laboratories undertook a pilot sequencing project to gain insight into the genome of Paramecium. Plasmid-end sequencing of DNA fragments from the somatic nucleus together with similarity searches identified 722 potential protein-coding genes. High gene density and uniform small intron size make random sequencing of somatic chromosomes a cost-effective strategy for gene discovery in this organism.

Transposable element-mediated enhancement of gene expression in Saccharomyces cerevisiae involves sequence-specific binding of a trans-acting factor.

In our studies on the regulation of adjacent-gene expression by Ty sequences, we demonstrated that a single-base-pair change (T-A----C-G) in the epsilon sequence of Ty917-derived elements is primarily responsible for enhancement of beta-galactosidase expression from lacZ fusion plasmids. Using an electrophoretic gel mobility assay, we showed that the same base pair transition is required for binding of a trans-acting factor, TyBF, from crude cell extracts in vitro. We identified the site of TyBF binding and determined the guanine nucleotide contact sites required for TyBF interaction. We propose that TyBF binding to cis-acting Ty2 sequences activates adjacent-gene transcription.

Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization.

Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro. Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo. Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect. Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position. Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae.

Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae. These plasmids transform S. cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells. One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA. The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S. cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus. Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S. cerevisiae 2-microns circle plasmid or with ars1, an S. cerevisiae chromosomal replicator.

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

Program Director Perceptions of Proficiency in the Core Entrustable Professional Activities.

BACKGROUND: The Association of American Medical Colleges describes 13 core entrustable professional activities (EPAs) that every graduating medical student should be expected to perform proficiently on day 1 of residency, regardless of chosen specialty. Studies have shown wide variability in program director (PD) confidence in interns' abilities to perform these core EPAs. Little is known regarding comparison of United States Medical Licensing Examination (USMLE) scores with proficiency in EPAs. OBJECTIVE: We determined if PDs from a large health system felt confident in their postgraduate year 1 residents' abilities to perform the 13 core EPAs, and compared perceived EPA proficiency with USMLE Step 1 and Step 2 scores. METHODS: The PDs were asked to rate their residents' proficiency in each EPA and to provide residents' USMLE scores. Timing coincided with the reporting period for resident milestones. RESULTS: Surveys were completed on 204 of 328 residents (62%). PDs reported that 69% of residents (140 of 204) were prepared for EPA 4 (orders/prescriptions), 61% (117 of 192) for EPA 7 (form clinical questions), 68% (135 of 198) for EPA 8 (handovers), 63% (116 of 185) for EPA 11 (consent), and 38% (49 of 129) for EPA 13 (patient safety). EPA ratings and USMLE 1 and 2 were negatively correlated (r(101) = -0.23, P = .031). CONCLUSIONS: PDs felt that a significant percentage of residents were not adequately prepared in order writing, forming clinical questions, handoffs, informed consent, and promoting a culture of patient safety. We found no positive association between USMLE scores and EPA ratings.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila.

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47-81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

The genomic structure of SYCP3, a meiosis-specific gene encoding a protein of the chromosome core.

SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.

An enrichment for temperature-sensitive mutants in Tetrahymena thermophila.

The parameters for the killing of Tetrahymena by 5-bromodeoxyuridine(BUdR) and near-ultraviolet light have been determined. Significant preferential killing by UV of cells that have incorporated BUdR was obtained when the cells were irradiated in a nonnutrient buffer. UV alone was found to be toxic to cells irradiated in growth medium. Mutants defective in division at a restrictive temperature were isolated from mutagenized cultures that had been treated with BUdR and UV and from mutagenized cultures that had no such treatment. Results indicate that the number of temperature sensitive (ts) growth mutants can be increase five to six times using the BUdR/UV treatment. Data are presented that indicate differences in the frequency of occurrence of various types of ts mutants, with and without enrichment. A mutant that immediately stopped macromolecular synthesis and cell division upon being placed at the restrictive temperature was more resistant to BUdR/UV treatment than wild type by 1000-fold. Using the above techniques, BUdR-resistant mutants altered in the phosphorylation of thymidine have been isolated.

Synaptonemal complexes from DNase-treated rat pachytene chromosomes contain (GT)n and LINE/SINE sequences.

Purified chromosome cores (synaptonemal complexes) of rat pachytene chromosomes, from which the chromatin is removed by extensive DNase II digestion, retain a residual class of DNA, presumably the bases of chromatin loops. This synaptonemal complex-associated DNA, isolated by proteinase digestion and phenol extraction of purified DNase-treated synaptonemal complexes, and cloned in plasmid vector pEMBL18, has a length distribution of 50-500 bp. From a library of these fragments, 21 fragments were sequenced. Present in this sample are short 40-200-bp segments with greater than 80% identity to "long" and "short" interspersed repeated elements (LINE/SINEs), an excess of GT/CA tandem repeats and a number of unidentified sequences. The LINE/SINE segments may play a role in homology vs. nonhomology recognition during meiosis and the alternating purine-pyrimidine sequences have been implicated in genetic recombination. Their enrichment in synaptonemal complexes may be related to the synapsis and recombination functions of meiosis.