bluuebunny/crossref_metadata_2025 huggingface dataset:
O63: POST-OPERATIVE ACUTE KIDNEY INJURY IN PATIENTS UNDERGOING MAJOR GASTROINTESTINAL SURGERY— AN OBSERVATIONAL COHORT STUDY AND EXTERNAL VALIDATION OF A PROGNOSTIC MODEL
Authors:   Collaborative STARSurg
Published: April 2021

Introduction

Acute kidney injury (AKI) is well-recognised as a significant cause of morbidity and mortality. Due to limited evidence on the longer-term implications, this study aimed to explore the association of postoperative AKI one-year survival and renal function in patients undergoing major gastrointestinal and liver surgery.

Method

Patients undergoing major gastrointestinal surgery in the prospective Outcomes of Kidney Injury after Surgery (OAKS) study across UK and Ireland were followed up at one-year postoperatively. The primary outcome was survival at 1-year and secondary outcomes included the composite “Major Adverse Kidney Events” outcome at day 365 (MAKE-365), with respective multivariable Cox-regression and logistic regression analysis performed.

Result

Of 62.2% of OAKS patients (n=3,575/5,745) with 1-year follow-up, there were no significant differences compared to those without follow-up. Among the follow-up cohort, 8.0% (n=269) patients died. On univariate analysis, patients experiencing 7-day postoperative AKI had a significantly higher hazard of death between 30 to 365 days postoperatively (HR: 2.10, 95% CI: 1.50-2.94, p<0.001) compared to patients who did not. This persisted on multivariable Cox-regression (HR: 1.67, 95% CI: 1.17-2.40, p=0.005). Furthermore, 9.1% (n=305) patients met the MAKE-365 endpoint. Multilevel logistic regression analysis demonstrated that the MAKE-365 endpoint was independently associated with both stage 1 (OR: 1.78, 95% CI: 1.22-2.61, p=0.003) and stage 2-3 7-day postoperative AKI (OR: 6.13, 95% CI: 3.97-9.45, p<0.001).

Conclusion

Post-operative AKI is associated with significantly higher rate of 1-year mortality and MAKE-365 endpoints. Improved monitoring of these patients may be warranted to identify and facilitate potential avenues for intervention

Take-home message

Post-operative AKI is associated with significantly higher rate of 1-year mortality. Hence, early detection and improved monitoring of patients with AKI with improve long-term outcomes of these patients.
O72: SECONDARY PREVENTION OF CARDIOVASCULAR DISEASE IN PERIPHERAL ARTERY DISEASE PATIENTS
Authors: Rashid ST , Bangee S
Published: April 2021

Introduction

Peripheral artery disease (PAD) is associated with significant cardiovascular morbidity and mortality which can be reduced by the treatment of atherosclerotic risk factors. Recent research demonstrates that novel drugs can significantly enhance PAD outcomes. However, the uptake of these drugs is uncertain. We wished to audit current atherosclerotic management in PAD patients as well as their eligibility for newer drugs - PCSK 9 inhibitors, SGLT2 inhibitors, and DOAC drugs – in accordance with relevant guidelines and trials.

Method

100 PAD inpatients and outpatients seen at a teaching hospital in the UK within the month of March 2019 were assessed. Medications from PAD patients were recorded from hospital records and patient histories were assessed for secondary prevention of cardiovascular disease - lipids, blood pressure, diabetic control, and atherothrombotic prevention.

Result

showed that compliance with NICE guidelines had a wide range – from 19% of outpatients on correct lipid control to 89% of inpatients on optimal atherothrombotic management. Based on the best evidence of eligibility: 0 of 41 eligible patients were taking PCSK9 Inhibitors, 1 out of the 71 was on an SGLT 2 inhibitor and 6 of 98 patients were on Rivaroxaban.

Conclusion

This audit shows adherence to NICE guidelines is variable for secondary prevention of atherosclerosis in PAD patients. Furthermore, very few novel treatments had been prescribed for suitable patients. Overall, prescribing for PAD risk factor management can be significantly improved. A solution could be dedicating risk factor clinics led by nurses, pharmacists or physicians.

Take-home message

Steps need to be taken to prevent cardiovascular disease in peripheral arterial disease patients. Steps include improvements in the following of guidelines, exploration of novel treatments, and the introduction of special risk factor clinics focused on improving the prognosis of the patients.
O80: GREMLIN-1 PROMOTES TUMOURIGENESIS AND METASTASIS IN HER2 POSITIVE BREAST CANCER
Authors: Zabkiewicz C , Ye L , Hargest R
Published: April 2021

Introduction

HER2 over-expression denotes poor prognosis in breast cancers.Bone morphogenetic protein(BMP) signalling is known to interact with EGF signalling, co-regulating breast cancer progression.BMP antagonist Gremlin-1 may influence breast cancer disease progression, but this remains unexplored in HER2 positive breast cancers.

Method

GREM1 and HER2 expression, and clinical outcomes were examined in clinical cohorts.GREM1 overexpression or pEF control plasmid were transduced into BT474 HER2+breast cancer cells. In vitro function tests using BT474 pEF and BT474GREM1cells include 2D/3D growth, migration, and expression of epithelial to mesenchymal transition(EMT)markers. Signalling cascades were examined in BT474 treated with RhGremlin-1. In vivo, BALB/c nude mice underwent either mammary injection or intra-cardiac injection of BT474pEF or BT474GREM1 cells and disease burden assessed.

Result

GREM1 expression correlates with HER2 in breast tumours(p=0.03) and is higher in metastatic HER2 positive cancers (p = 0.04). HER2 positive patients with high GREM1 have poor survival(p = 0.0002). BT474GREM1cells have up-regulated markers of EMT compared to control. BT474 RhGremlin-1 treated cells have active AKT pathway signalling, independent of BMP signalling. In vitro,  BT474GREM1cells significantly proliferate and migrate compared to control(p<0.05 and p < 0.001).This is confirmed in vivo,  BT474GREM1 mice grew significantly larger mammary tumours(p<0.05) and had more PETCT metastatic hotspots.

Conclusion

Gremlin-1 is correlated with poor outcomes in HER2 patients and promotes breast cancer cellular growth, migration and metastasis.Gremlin-1 is a novel area of research with potential as a prognostic biomarker and therapeutic target for personalised, effective, breast cancer outcomes.

Take-home message

BMP antagonists are gaining interest for their potential in breast cancer prognosis and therapeutics.This novel area of research shows BMP antagonist Gremlin-1 is of importance in HER2 positive breast cancers.

DRAGONS DEN
P4: EXTERNAL VALIDATION OF THE OUTCOMES AFTER KIDNEY INJURY IN SURGERY (OAKS) PROGNOSTIC SCORE TO PREDICT POSTOPERATIVE ACUTE KIDNEY INJURY IN PATIENTS UNDERGOING MAJOR ELECTIVE COLORECTAL SURGERY
Authors:   STARSurg Collaborative, EuroSurg Collaborative
Published: April 2021

Background

Postoperative Acute Kidney Injury (AKI) is associated with significant morbidity and mortality following major gastrointestinal surgery. The Outcomes After Kidney injury in Surgery (OAKS) prognostic score risk stratifies patients undergoing major gastrointestinal surgeries for 7-day postoperative acute kidney injury (AKI). This study aimed to perform external validation of an international elective colorectal cohort.

Method

The Ileus MAnagement INternational (IMAGINE) audit (January to April 2018) included consecutive adults undergoing elective colorectal resection or stoma reversal across Europe and Australasia. Multivariate logistic regression was performed using data on 7-day AKI and the OAKS prognostic variables (age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery, and preoperative use of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers). Discrimination and calibration (Hosmer–Lemeshow test) were assessed on prediction of patients at high-risk (>20%) of postoperative AKI.

Result

Of 4046 patients included across 338 centres, 13.4% (n=542) developed 7-day AKI. The model discrimination was 0.67 (95% CI = 0.65-0.70), identifying high-risk patients with low sensitivity (0.28, 95% CI: 0.25-0.32) but high specificity (0.90, 95% CI = 0.89-0.91). Furthermore, the model demonstrated good calibration (p=0.518).

Conclusion

Discrimination of the OAKS score for patients at high-risk of postoperative AKI in this cohort is good, and remains consistent with the derivation cohort. High-risk patients identified may represent a feasible target for interventions aimed at mitigating AKI.

Take-home message

Prognostic risk scores may be helpful in predicting risk of postoperative acute kidney injury in patients undergoing major abdominal surgery.
P7: ACUTE KIDNEY INJURY AFTER ELECTIVE COLORECTAL SURGERY: RESULTS OF A NATIONAL CLOSED-LOOP AUDIT
Authors:   STARSurg Collaborative, EuroSurg Collaborative
Published: April 2021

Introduction

The aim of this study was to re-audit the rates of acute kidney injury (AKI) after elective colorectal surgery, following local presentations of results.

Method

Outcomes After Kidney injury in Surgery (OAKS) and Ileus Management International (IMAGINE), were prospective multicentre audits on consecutive elective colorectal resections, in the UK and Ireland. These were performed over 3-month periods in 2015 and 2018 respectively. During the interim period, results were presented at participating centres to stimulate local quality improvement initiatives. Risk-adjusted 7-day postoperative AKI rates were calculated through multilevel logistic regression based on the OAKS prognostic score.

Result

Of the 4,917 patients included, 3,133 (63.7%) originated from OAKS and 1,784 (36.3%) from IMAGINE. On univariate analysis, there was no significant difference (p=0.737) in the 7-day AKI rate between OAKS (n=346, 11.8%) and IMAGINE (n=205, 11.5%). However, the risk-adjusted AKI rate in IMAGINE was significantly lower compared to OAKS (-1.8%, 95% CI: -2.3% to -1.3%, p<0.001). Of 47 centres (40.1%) with a recorded local presentation, there was no significant difference in the subsequent AKI rate in IMAGINE (-0.7%, -2.0% to 0.6%, p=0.278).

Conclusion

Rates of AKI after elective colorectal surgery significantly reduced on re-audit. However, this may be related to increased awareness from participation or national quality improvement initiatives, rather than local presentation of results. Abbrev. AKI - Acute Kidney Injury, OAKS - Outcomes After Kidney injury in Surgery, IMAGINE - Ileus Management International

Take-home message

Risk-adjusted AKI rates significantly reduced on re-audit, however, this was most likely due to factors separate from the local presentation of initial results.
P8: A COMPARATIVE EVALUATION OF INTENSIVE OUT-OF-TERM AND CONVENTIONAL TERM-TIME MENTORING SCHEMES IN PREPARING MEDICAL STUDENTS FOR A CAREER IN SURGERY
Authors: Sawhney R , Ingram J , Smith J
Published: April 2021

Introduction

Mentoring schemes have been established to mitigate these concerns and improve interest in surgery among medical students; however, there is a paucity of research on whether conventional term-time (CTT) mentoring or more intensive out-of-term (IOT) mentoring is more meaningful in promoting surgical careers.

Method

Most surgical societies in the UK offer CTT schemes (ca. one-to-three mentoring during the academic year) to their members. In addition to this, a student-led surgical society piloted an IOT scheme (one-to-one mentoring during the summer break). Students completed a ten-point questionnaire at the end of the academic year evaluating the scheme(s) they participated in.

Result

Of the 35 students that completed a questionnaire, 74% participated in either CTT or IOT (1:1), and 26% participated in both. Students that participated in one scheme considered that scheme to be superior in increasing interest in surgery (CTT 69%; IOT 79%), while students that participated in both favoured IOT (78%). With regards to increasing preparedness for surgical careers, IOT was universally preferred. According to previously determined levels of engagement, maximum engagement was achieved by 61.5% of students in a single week of IOT, compared to only 7.7% of students after a full academic year of CTT.

Conclusion

IOT outperforms CTT with regards to boosting interest in surgery and preparedness for surgical careers. This suggests that introduction of IOT schemes nationally may be a valuable advance in the future of surgical mentoring.

Take-home message

Adopting an intensive out-of-term mentoring scheme has shown a positive outcome in terms of both increasing preparedness for, and interest in, surgical careers. This suggests implementing such time intensive schemes nationally could be a sensible future-step in surgical mentoring.
P13: CHARACTERISATION OF THE TUMOUR MICROENVIRONMENT IN MESENCHYMAL GASTRIC TUMOURS USING SINGLE-CELL RNA SEQUENCING
Authors: Harrington J , Lloyd M , Mabrouk N , Walker R , Grace B , Underwood T
Published: April 2021

Introduction

Gastric mesenchymal tumours are a rare group of neoplasms, which include gastrointestinal stromal tumours (GISTs) and leiomyomas. To date, there is limited information on the tumour microenvironment (TME) in these neoplasms, despite the TME widely known to influence the hallmarks of cancer. In this study we used single cell RNA sequencing (scRNAseq) to profile individual cells of the TME in GIST and leiomyoma.

Method

The two gastric mesenchymal tumours and two normal gastric samples were analysed using DropSeq, where single cell transcriptomes are captured onto barcoded beads using a microfluidic device before next generation sequencing. For comparison, we performed bulk RNA-sequencing and CIBERSORT to estimate the abundance of 22 immune cell populations. Furthermore, we used immunohistochemistry to elucidate the presence and location of several immune cells.

Result

Both neoplasms had diverse immune and stromal cell populations with a greater proportion of macrophages but less B cells than normal gastric tissue. ScRNAseq was able to identify subpopulations of B cells and T cells not detected with CIBERSORT. Interstitial cells of cajal, believed to be the pre-cursor to GISTs, were observed through scRNAseq and confirmed through immunohistochemistry.

Conclusion

To our knowledge, this is the first study to utilise scRNAseq on GISTs and leiomyomas, which enabled characterisation of the TME at a cellular level. Using this platform in future studies will enable better characterisation of the TME and may inform the discovery of therapeutic targets.

Take-home message

Single cell RNA sequencing enables the ability to explore the tumour microenvironment of mesenchymal tumours at an enhanced resolution, paving the way for potential future therapeutic targets.
P20: DOES PRE-OP MRI FOLLOWING NEOADJUVANT LONG COURSE CHEMO-RADIOTHERAPY IN RECTAL CANCER AFFECT R0 RESECTION RATE?
Authors: Borucki JP , Jones KL , Stearns AT
Published: April 2021

Introduction

Neoadjuvant long course chemo-radiotherapy (LCCR) is the standard of care for locally advanced rectal cancer. In June 2013 this institution implemented preoperative MRI post LCCR in an attempt to improve the R0 resection rate. The aims of this projects were to audit the compliance with the new policy and to assess whether it had an impact on the R0 resection rate.

Method

All patients who underwent resection after LCCR between June 2013 and February 2017 had their clinical notes audited to assess compliance with the standard ‘all patients who have undergone LCCR should receive MRI imaging of the rectum unless clinically contraindicated’. To assess effectiveness of the intervention the R0 resection rate of patients who had received the preoperative MRI was compared to those who had not between the period January 2008 and February 2017. The data was entered and stored onto a larger departmental database so that it could be used in future for other projects.

Result

33 out of 88 (37.5%) of eligible patients received post LCCR MRI rectum prior to resection although a further two patients had a preoperative CT scan. The R0 resection rate in patients who had pre-operative MRI was 88% compared to 89% in those who did not (p=1).

Conclusion

The rate of patients who received pre-operative MRI following LCCR was 37.5%. Whilst this shows significant room for improvement, data collected regarding effectiveness of the intervention shows that it makes no difference to R0 resection rate.

Take-home message

Pre-op MRI following LCCR does not improve R0 resection rate in locally advanced rectal cancer.
P23: ENHANCED RECOVERY PROGRAMME – DOES PREOPERATIVE EDUCATION REDUCE LENGTH OF HOSPITAL STAY (LOS) IN PRIMARY HIP & KNEE ARTHROPLASTY
Authors: Ahmad M , Rahman H , Awan N
Published: April 2021

Introduction

Total knee and hip arthroplasty significantly improve quality of life in people suffering from end-stage hip & knee arthritides. Enhanced recovery programs have shown improved outcomes following elective arthroplasty by decreasing patients anxiety, postoperative pain and reducing the length of hospital stay (LOS). The aim of our study is to evaluate the impact of preoperative education programs on length of hospital stay in patients undergoing hip & knee arthroplasty.

Method

Retrospective data was collected from a consecutive series of 520 patients' charts and Irish National Orthopedic Register (INOR) who underwent Hip or Knee arthroplasty from January 1, 2018, to December 31, 2018, in Our Lady Hospital, Navan. The length of stay study compared two hundred and twenty-six patients in the Educational Group (EG) who attended the Hip & Knee School (Preoperative Education Class) with 294 patients in the Conventional Group (CG) who did not attend the hip and knee educational programme.

Result

We identified that Length of Hospital Stay decreased to 5.2 days in Educational Group from 5.5 days in Conventional Group (P-value equals to 0.26, statistically insignificant, t = 1.1093 df = 518), with a mean difference of only 0.3 days (95% confidence interval).

Conclusion

Preoperative education does not reduce the length of hospital stay in primary hip & knee arthroplasty.

Take-home message

Preoperative education has no role in reducing hospital stay in Arthroplasty patients.
P28: LAPAROSCOPIC VERSUS ULTRASOUND-GUIDED TRANSVERSUS ABDOMINIS PLANE BLOCK IN LAPAROSCOPIC COLORECTAL SURGERY - A SYSTEMATIC REVIEW AND META-ANALYSIS
Authors: Copperthwaite A , Sahebally S , Devane L , McCawley N , Burke J , McNamara D
Published: April 2021

Introduction

A multimodal approach to peri-operative analgesia represents a cornerstone of enhanced recovery programmes in minimally invasive colorectal surgery. Transversus abdominis plane (TAP) block is an emerging anaesthetic technique that may reduce postoperative opiod analgesic requirements. It can be performed laparoscopically or percutaneously. We performed a systematic review and meta-analysis to compare the effectiveness of laparoscopic versus ultrasound-guided TAP block in laparoscopic colorectal surgery.

Method

PubMed, EMBASE and Cochrane databases were searched for relevant articles from inception until November 2019. All studies that compared laparoscopic (LTB) versus ultrasound-guided (UTB) TAP blocks in laparoscopic colorectal resections were included. The primary outcome measure was narcotic consumption at 24 hours postoperatively, while secondary outcomes included pain scores, operative time, postoperative nausea and vomitting (PONV) and complication rates. Random effects models were used to calculate pooled effect size estimates.

Result

Three randomised controlled trials and one retrospective study were included capturing 309 patients. On random effects analysis, there were no significant differences in narcotic consumption between LTB and UTB (Mean difference -8.12 mg, 95% CI=-22.13 to 5.89, p=0.26). LTB was associated with significantly lower pain scores (Mean difference -0.49, 95% CI=-0.96 to -0.02, p=0.04). There were no differences in operative time (Mean difference -6.67 mins, 95% CI=-29.4 to 16.06, p=0.57), PONV (OR=0.97, 95% CI=0.36 to 2.65, p=0.96) or complication (OR=1.30, 95% CI=0.64 to 2.64, p=0.47) rates.

Conclusion

LTB is associated with significantly less pain at 24 hours postoperatively but similar narcotic usage, PONV, operative time and complication rates, compared to UTB.

Take-home message

Laparoscopic transversus abdominis plane block is shown to leave patients with less pain at 24 hours post-op compared to ultrasound guided methods in colorectal patients.
Achievement Rank Affects Performance and Major Choices in College
Authors: Elsner Benjamin , Isphording Ingo E , Zölitz Ulf
Published: April 2021

In this paper we study how a student's ordinal rank in a peer group affects performance and specialisation choices in university. By exploiting data with repeated random assignment of students to teaching sections, we find that a higher rank increases performance and the probability of choosing related follow-up courses and majors. We document two types of dynamic effect. First, earlier ranks are less important than later ranks. Second, responses to rank changes are asymmetric: improvements in rank raise performance, while decreases in rank have no effect. Rank effects partially operate through students’ expectations about future grades.
Probable Mechanisms Underlying Interallelic Complementation and Temperature-Sensitivity of Mutations at the shibire Locus of Drosophila melanogaster
Authors: Grant Deanna , Unadkat Shilpa , Katzen Alisa , Krishnan K S , Ramaswami Mani
Published: April 2021

The shibire locus of Drosophila melanogaster encodes dynamin, a GTPase required for the fission of endocytic vesicles from plasma membrane. Biochemical studies indicate that mammalian dynamin is part of a complex containing multiple dynamin subunits and other polypeptides. To gain insight into sequences of dynamin critical for its function, we have characterized in detail a collection of conditional and lethal shi alleles. We describe a probable null allele of shi and show that its properties are distinct from those of two classes of lethal alleles (termed I and II) that show intergroup, interallelic complementation. Sequenced class I alleles, which display dominant properties, carry missense mutations in conserved residues in the GTPase domain of dynamin. In contrast, the sequenced class II alleles, which appear completely recessive, carry missense mutations in conserved residues of a previously uncharacterized “middle domain” that lies adjacent to the GTPase region. These data suggest that critical interactions mediated by this middle domain are severely affected by the class II lethal mutations; thus, the mutant sequences should be very useful for confirming the in vivo relevance of interactions observed in vitro. Viable heteroallelic combinations of shi lethals show rapid and reversible temperature-sensitive paralytic phenotypes hitherto only described for the ts alleles of shi. When taken together with the molecular analysis of shi mutations, these observations suggest that the GTPase domain of dynamin carries an intrinsically temperature-sensitive activity: hypomorphic mutations that reduce this activity at low temperatures result in conditional temperature-sensitive phenotype. These observations explain why screens for conditional paralytic mutants in Drosophila inevitably recover ts alleles of shi at high frequencies.
Topoisomerase I Is Essential in Cryptococcus neoformans: Role in Pathobiology and as an Antifungal Target
Authors: Del Poeta Maurizio , Toffaletti Dena L , Rude Thomas H , Dykstra Christine C , Heitman Joseph , Perfect John R
Published: April 2021

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, γ-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.
The Aspergillus nidulans uvsB Gene Encodes an ATM-Related Kinase Required for Multiple Facets of the DNA Damage Response
Authors: Hofmann Amy F , Harris Steven D
Published: April 2021

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.
Identification of a Mutant Locus by Noncomplementation of a Transposon Insertion Library in Saccharomyces cerevisiae
Authors: Wiatrowski Heather A , Carlson Marian
Published: April 2021

We describe a new approach for identifying the gene corresponding to a mutation in Saccharomyces cerevisiae. A library of mTn-lacZ/LEU2 insertions is tested for failure to complement the mutation, and the noncomplementing insertion is used to obtain sequence. This approach offers an alternative to cloning by complementation with a plasmid library.
Genetic Analysis of a Synaptic Calcium Channel in Drosophila: Intragenic Modifiers of a Temperature-Sensitive Paralytic Mutant of cacophony
Authors: Brooks I M , Felling R , Kawasaki F , Ordway R W
Published: April 2021

Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel α1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cacTS2, indicated cac encodes a primary calcium channel α1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cacTS2 paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cacTS2, including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel α1 subunit function in neurotransmitter release.
Evolution of MHC Class II Eβ Diversity Within the Genus Peromyscus
Authors: Richman Adam D , Herrera L Gerardo , Nash Deanna
Published: April 2021

Progress in understanding the evolution of variation at the MHC has been slowed by an inability to assess the relative roles of mutation vs. intragenic recombination in contributing to observed polymorphism. Recent theoretical advances now permit a quantitative treatment of the problem, with the result that the amount of recombination is at least an order of magnitude greater than that of mutation in the history of class II genes. We suggest that this insight allows progress in evaluating the importance of other factors affecting the evolution of the MHC. We investigated the evolution of MHC class II Eβ sequence diversity in the genus Peromyscus. We find evidence for extensive recombination in the history of these sequences. Nevertheless, it appears that intragenic recombination alone is insufficient to account for evolution of MHC diversity in Peromyscus. Significant differences in silent variation among subgenera arose over a relatively short period of time, with little subsequent change. We argue that these observations are consistent with the effects of historical population bottleneck(s). Population restrictions may explain general features of MHC evolution, including the large amount of recombination in the history of MHC genes, because intragenic recombination may efficiently regenerate allelic polymorphism following a population constriction.
Gene Silencing Triggered by Non-LTR Retrotransposons in the Female Germline of Drosophila melanogaster
Authors: Robin Stéphanie , Chambeyron Séverine , Bucheton Alain , Busseau Isabelle
Published: April 2021

Several studies have recently shown that the activity of some eukaryotic transposable elements is sensitive to the presence of homologous transgenes, suggesting the involvement of homology-dependent genesilencing mechanisms in their regulation. Here we provide data indicating that two non-LTR retrotransposons of Drosophila melanogaster are themselves natural triggers of homology-dependent gene silencing. We show that, in the female germline of D. melanogaster, fragments from the R1 or from the I retrotransposons can mediate silencing of chimeric transcription units into which they are inserted. This silencing is probably mediated by sequence identity with endogenous copies of the retrotransposons because it does not occur with a fragment from the divergent R1 elements of Bombyx mori, and, when a fragment of I is used, it occurs only in females containing functional copies of the I element. This silencing is not accompanied by cosuppression of the endogenous gene homologous to the chimeric transcription unit, which contrasts to some other silencing mechanisms in Drosophila. These observations suggest that in the female germline of D. melanogaster the R1 and I retrotransposons may self-regulate their own activity and their copy number by triggering homology-dependent gene silencing.
The Drosophila roX1 RNA Gene Can Overcome Silent Chromatin by Recruiting the Male-Specific Lethal Dosage Compensation Complex
Authors: Kelley Richard L , Kuroda Mitzi I
Published: April 2021

The Drosophila MSL complex consists of at least six proteins and two noncoding roX RNAs that mediate dosage compensation. It acts to remodel the male's X chromatin by covalently modifying the amino terminal tails of histones. The roX1 and roX2 genes are thought to be nucleation sites for assembly and spreading of MSL complexes into surrounding chromatin where they roughly double the rates of transcription. We generated many transgenic stocks in which the roX1 gene was moved from its normal location on the X to new autosomal sites. Approximately 10% of such lines displayed unusual sexually dimorphic expression patterns of the transgene's mini-white eye-color marker. Males often displayed striking mosaic pigmentation patterns similar to those seen in position-effect variegation and yet most inserts were in euchromatic locations. In many of these stocks, female mini-white expression was very low or absent. The male-specific activation of mini-white depended upon the MSL complex. We propose that these transgenes are inserted in several different types of repressive chromatin environments that inhibit mini-white expression. Males are able to overcome this silencing through the action of the MSL complex spreading from the roX1 gene and remodeling the local chromatin to allow transcription. The potency with which an ectopic MSL complex overcomes silent chromatin suggests that its normal action on the X must be under strict regulation.
Phenotypic Analysis of Separation-of-Function Alleles of MEI-41, Drosophila ATM/ATR
Authors: Laurençon Anne , Purdy Amanda , Sekelsky Jeff , Hawley R Scott , Su Tin Tin
Published: April 2021

ATM/ATR kinases act as signal transducers in eukaryotic DNA damage and replication checkpoints. Mutations in ATM/ATR homologs have pleiotropic effects that range from sterility to increased killing by genotoxins in humans, mice, and Drosophila. Here we report the generation of a null allele of mei-41, Drosophila ATM/ATR homolog, and the use of it to document a semidominant effect on a larval mitotic checkpoint and methyl methanesulfonate (MMS) sensitivity. We also tested the role of mei-41 in a recently characterized checkpoint that delays metaphase/anaphase transition after DNA damage in cellular embryos. We then compare five existing mei-41 alleles to the null with respect to known phenotypes (female sterility, cell cycle checkpoints, and MMS resistance). We find that not all phenotypes are affected equally by each allele, i.e., the functions of MEI-41 in ensuring fertility, cell cycle regulation, and resistance to genotoxins are genetically separable. We propose that MEI-41 acts not in a single rigid signal transduction pathway, but in multiple molecular contexts to carry out its many functions. Sequence analysis identified mutations, which, for most alleles, fall in the poorly characterized region outside the kinase domain; this allowed us to tentatively identify additional functional domains of MEI-41 that could be subjected to future structure-function studies of this key molecule.
Change of the Heterogametic Sex From Male to Female in the Frog
Authors: Ogata M , Ohtani H , Igarashi T , Hasegawa Y , Ichikawa Y , Miura I
Published: April 2021

Two different types of sex chromosomes, XX/XY and ZZ/ZW, exist in the Japanese frog Rana rugosa. They are separated in two local forms that share a common origin in hybridization between the other two forms (West Japan and Kanto) with male heterogametic sex determination and homomorphic sex chromosomes. In this study, to find out how the different types of sex chromosomes differentiated, particularly the evolutionary reason for the heterogametic sex change from male to female, we performed artificial crossings between the West Japan and Kanto forms and mitochondrial 12S rRNA gene sequence analysis. The crossing results showed male bias using mother frogs with West Japan cytoplasm and female bias using those with Kanto cytoplasm. The mitochondrial genes of ZZ/ZW and XX/XY forms, respectively, were similar in sequence to those of the West Japan and Kanto forms. These results suggest that in the primary ZZ/ZW form, the West Japan strain was maternal and thus male bias was caused by the introgression of the Kanto strain while in the primary XX/XY form and vice versa. We therefore hypothesize that sex ratio bias according to the maternal origin of the hybrid population was a trigger for the sex chromosome differentiation and the change of heterogametic sex.
A New Genetic Locus Controlling Growth and Proliferation in Drosophila melanogaster
Authors: Raisin Sophie , Pantalacci Sophie , Breittmayer Jean-Philippe , Léopold Pierre
Published: April 2021

Multicellular organisms grow through both proliferation and growth of their individual cells. We have conducted a P-element-based misexpression screen for genes whose upregulation alters wing disc growth during development. One particular group of four P elements, all inserted at cytological location 61C7-8, exhibited specific overgrowth upon misexpression in proliferating imaginal tissues. Clonal analysis revealed that upon misexpression, cell number was increased but cell size was not affected, indicating that cell growth and proliferation were induced in a coordinate manner. Loss of function at the locus produced small flies with reduced cell number, consistent with the presence of a gene encoding a positive growth regulator. We characterized a new transcription unit initiating in a region adjacent to the P insertions, which generated a complex series of polyadenylated transcripts. Although these RNAs were induced in response to misexpression, none was sufficient by itself to recapitulate overgrowth when overexpressed. This suggested either that a particular combination of these transcripts was necessary or that other sequences are involved.
Mutation in Intron 6 of the Hamster Mitf Gene Leads to Skipping of the Subsequent Exon and Creates a Novel Animal Model for the Human Waardenburg Syndrome Type II
Authors: Graw Jochen , Pretsch Walter , Löster Jana
Published: April 2021

In the course of analysis of ENU-induced mutations in Syrian hamsters, a novel dominant anophthalmic white mutant (WhV203) with hearing loss was recovered. Because of this phenotype and a close linkage to the Tpi gene, the Mitf gene was considered as a candidate gene. In the Mitf cDNA, a deletion of 76 bp covering the entire exon 7 was detected. Further molecular analysis revealed a T → A exchange 16 bp upstream of the end of intron 6, leading to skipping of exon 7. These 16 bp at the end of intron 6 are identical in hamster, rat, mouse, and humans, indicating high conservation during evolution and a functional importance in splicing. Since the loss of exon 7 changes the open reading frame of the MITF transcript, translation will be stopped after 10 new amino acids. The truncated protein is predicted to contain only a part of the basic region and will miss the two helical domains and the leucine zipper. The WhV203 mutation in the Syrian hamster affects the same functional domains of the Mitf transcription factor as the human R124X mutation, causing human Waardenburg syndrome type II. Therefore, the WhV203 hamster mutant provides a novel model for this particular syndrome.
Scaling of Mutational Effects in Models for Pleiotropy
Authors: Wingreen Ned S , Miller Jonathan , Cox Edward C
Published: April 2021

Mutation-selection models provide a framework to relate the parameters of microevolution to properties of populations. Like all models, these must be subject to test and refinement in light of experiments. The standard mutation-selection model assumes that the effects of a pleiotropic mutation on different characters are uncorrelated. As a consequence of this assumption, mutations of small overall effect are suppressed. For strong enough pleiotropy, the result is a nonvanishing fraction of a population with the “perfect” phenotype. However, experiments on microorganisms and experiments on protein structure and function contradict the assumptions of the standard model, and Kimura’s observations of heterogeneity within populations contradict its conclusions. Guided by these observations, we present an alternative model for pleiotropic mutations. The new model allows mutations of small overall effect and thus eliminates the finite fraction of the population with the perfect phenotype.
Effect of Recombination on the Accuracy of the Likelihood Method for Detecting Positive Selection at Amino Acid Sites
Authors: Anisimova Maria , Nielsen Rasmus , Yang Ziheng
Published: April 2021

Maximum-likelihood methods based on models of codon substitution accounting for heterogeneous selective pressures across sites have proved to be powerful in detecting positive selection in protein-coding DNA sequences. Those methods are phylogeny based and do not account for the effects of recombination. When recombination occurs, such as in population data, no unique tree topology can describe the evolutionary history of the whole sequence. This violation of assumptions raises serious concerns about the likelihood method for detecting positive selection. Here we use computer simulation to evaluate the reliability of the likelihood-ratio test (LRT) for positive selection in the presence of recombination. We examine three tests based on different models of variable selective pressures among sites. Sequences are simulated using a coalescent model with recombination and analyzed using codon-based likelihood models ignoring recombination. We find that the LRT is robust to low levels of recombination (with fewer than three recombination events in the history of a sample of 10 sequences). However, at higher levels of recombination, the type I error rate can be as high as 90%, especially when the null model in the LRT is unrealistic, and the test often mistakes recombination as evidence for positive selection. The test that compares the more realistic models M7 (β) against M8 (β and ω) is more robust to recombination, where the null model M7 allows the positive selection pressure to vary between 0 and 1 (and so does not account for positive selection), and the alternative model M8 allows an additional discrete class with ω= dN/dS that could be estimated to be >1 (and thus accounts for positive selection). Identification of sites under positive selection by the empirical Bayes method appears to be less affected than the LRT by recombination.
Functional Genomics Reveals Relationships Between the Retrovirus-Like Ty1 Element and Its Host Saccharomyces cerevisiae
Authors: Griffith Jacqulyn L , Coleman Laura E , Raymond Adam S , Goodson Summer G , Pittard William S , Tsui Circe , Devine Scott E
Published: April 2021

Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.
Dntf-2r, a Young Drosophila Retroposed Gene With Specific Male Expression Under Positive Darwinian Selection
Authors: Betrán Esther , Long Manyuan
Published: April 2021

A direct approach to investigating new gene origination is to examine recently evolved genes. We report a new gene in the Drosophila melanogaster subgroup, Drosophila nuclear transport factor-2-related (Dntf-2r). Its sequence features and phylogenetic distribution indicate that Dntf-2r is a retroposed functional gene and originated in the common ancestor of D. melanogaster, D. simulans, D. sechellia, and D. mauritiana, within the past 3-12 million years (MY). Dntf-2r evolved more rapidly than the parental gene, under positive Darwinian selection as revealed by the McDonald-Kreitman test and other evolutionary analyses. Comparative expression analysis shows that Dntf-2r is male specific whereas the parental gene, Dntf-2, is widely expressed in D. melanogaster. In agreement with its new expression pattern, the Dntf-2r putative promoter sequence is similar to the late testis promoter of β2-tubulin. We discuss the possibility that the action of positive selection in Dntf-2r is related to its putative male-specific functions.
Associations Between Inverted Repeats and the Structural Evolution of Bacterial Genomes
Authors: Achaz Guillaume , Coissac Eric , Netter Pierre , Rocha Eduardo P C
Published: April 2021

The stability of the structure of bacterial genomes is challenged by recombination events. Since major rearrangements (i.e., inversions) are thought to frequently operate by homologous recombination between inverted repeats, we analyzed the presence and distribution of such repeats in bacterial genomes and their relation to the conservation of chromosomal structure. First, we show that there is a strong underrepresentation of inverted repeats, relative to direct repeats, in most chromosomes, especially among the ones regarded as most stable. Second, we show that the avoidance of repeats is frequently associated with the stability of the genomes. Closely related genomes reported to differ in terms of stability are also found to differ in the number of inverted repeats. Third, when using replication strand bias as a proxy for genome stability, we find a significant negative correlation between this strand bias and the abundance of inverted repeats. Fourth, when measuring the recombining potential of inverted repeats and their eventual impact on different features of the chromosomal structure, we observe a tendency of repeats to be located in the chromosome in such a way that rearrangements produce a smaller strand switch and smaller asymmetries than expected by chance. Finally, we discuss the limitations of our analysis and the influence of factors such as the nature of repeats, e.g., transposases, or the differences in the recombination machinery among bacteria. These results shed light on the challenges imposed on the genome structure by the presence of inverted repeats.
Identification of Genes That Regulate a Left-Right Asymmetric Neuronal Migration in Caenorhabditis elegans
Authors: Ch’ng QueeLim , Williams Lisa , Lie Yung S , Sym Mary , Whangbo Jennifer , Kenyon Cynthia
Published: April 2021

In C. elegans, cells of the QL and QR neuroblast lineages migrate with left-right asymmetry; QL and its descendants migrate posteriorly whereas QR and its descendants migrate anteriorly. One key step in generating this asymmetry is the expression of the Hox gene mab-5 in the QL descendants but not in the QR descendants. This asymmetry appears to be coupled to the asymmetric polarizations and movements of QL and QR as they migrate and relies on an asymmetric response to an EGL-20/Wnt signal. To identify genes involved in these complex layers of regulation and to isolate targets of mab-5 that direct posterior migrations, we screened visually for mutants with cell migration defects in the QL and QR lineages. Here, we describe a set of new mutants (qid-5, qid-6, qid-7, and qid-8) that primarily disrupt the migrations of the QL descendants. Most of these mutants were defective in mab-5 expression in the QL lineage and might identify genes that interact directly or indirectly with the EGL-20/Wnt signaling pathway.