Heteroduplex DNA and meiotic recombination in Drosophila

Meiotic recombination gives rise to crossovers, which are required in most organisms for the faithful segregation of homologous chromosomes during meiotic cell division. Investigation of the details of this process has centered largely on studies in fungi; however, recent evidence suggests that a complete understanding will require this question to be approached in multiple model organisms. In this thesis, I report the development of tools for expanding the study of meiotic recombination in Drosophila melanogaster. I have combined an existing assay for the selection of rare recombination events with molecular techniques to allow the fine dissection of the structures of recombination events. I demonstrate the utility of this assay by using it to investigate recombination in a known meiotic mutant, mei-9. The results of this investigation provide evidence supporting a role for MEI-9 in resolving recombination intermediates to generate crossovers, and supporting a model in which noncrossovers are generated by multiple pathways. I also report the genetic characterization of the role of a partner protein of MEI-9, ERCC1. These results suggest that the functional protein complex for the generation of meiotic crossovers contains MEI-9, ERCC1, and a previously-characterized protein, MUS312. Additionally, I created a Drosophila mismatch repair mutant, Msh6, and used my newly-developed assay to investigate meiotic recombination events in this mutant. This constitutes the first investigation of meiotic mismatch repair in Drosophila. Results of this assay show that elimination of mismatch repair allows the recovery of unrepaired heteroduplex DNA with high efficiency. Characterization of the structure and arrangement of heteroduplex DNA is instrumental in the dissection of molecular models of meiotic recombination. The creation of a mutant in which heteroduplex DNA can be recovered in Drosophila does much to put the molecular characterization of meiotic recombination in this organism on a par with studies in fungi. Using this assay, I also show strong evidence for a "short-patch" repair pathway that acts in the absence of canonical mismatch repair in Drosophila. In this thesis, I demonstrate the importance and effectiveness of using Drosophila to further our understanding of the crucial process of meiotic recombination, and report the development of a number of tools that demonstrate the attractiveness of using this model organism for these studies

Meiotic Recombination in Drosophila Msh6 Mutants Yields Discontinuous Gene Conversion Tracts

Crossovers (COs) generated through meiotic recombination are important for the correct segregation of homologous chromosomes during meiosis. Several models describing the molecular mechanism of meiotic recombination have been proposed. These models differ in the arrangement of heteroduplex DNA (hDNA) in recombination intermediates. Heterologies in hDNA are usually repaired prior to the recovery of recombination products, thereby obscuring information about the arrangement of hDNA. To examine hDNA in meiotic recombination in Drosophila melanogaster, we sought to block hDNA repair by conducting recombination assays in a mutant defective in mismatch repair (MMR). We generated mutations in the MMR gene Msh6 and analyzed recombination between highly polymorphic homologous chromosomes. We found that hDNA often goes unrepaired during meiotic recombination in an Msh6 mutant, leading to high levels of postmeiotic segregation; however, hDNA and gene conversion tracts are frequently discontinuous, with multiple transitions between gene conversion, restoration, and unrepaired hDNA. We suggest that these discontinuities reflect the activity of a short-patch repair system that operates when canonical MMR is defective

Randomized trial of an education and support intervention to preventreadmission of patients with heart failure

AbstractObjectivesWe determined the effect of a targeted education and support intervention on the rate of readmission or death and hospital costs in patients with heart failure (HF).BackgroundDisease management programs for patients with HF including medical components may reduce readmissions by 40% or more, but the value of an intervention focused on education and support is not known.MethodsWe conducted a prospective, randomized trial of a formal education and support intervention on one-year readmission or mortality and costs of care for patients hospitalized with HF.ResultsAmong the 88 patients (44 intervention and 44 control) in the study, 25 patients (56.8%) in the intervention group and 36 patients (81.8%) in the control group had at least one readmission or died during one-year follow-up (relative risk = 0.69, 95% confidence interval [CI]: 0.52, 0.92; p = 0.01). The intervention was associated with a 39% decrease in the total number of readmissions (intervention group: 49 readmissions; control group: 80 readmissions, p = 0.06). After adjusting for clinical and demographic characteristics, the intervention group had a significantly lower risk of readmission compared with the control group (hazard ratio = 0.56, 95% CI: 0.32, 0.96; p = 0.03) and hospital readmission costs of $7,515 less per patient.ConclusionsA formal education and support intervention substantially reduced adverse clinical outcomes and costs for patients with HF

Calling by Concluding Sentinels: Coordinating Cooperation or Revealing Risk?

Efficient cooperation requires effective coordination of individual contributions to the cooperative behaviour. Most social birds and mammals involved in cooperation produce a range of vocalisations, which may be important in regulating both individual contributions and the combined group effort. Here we investigate the role of a specific call in regulating cooperative sentinel behaviour in pied babblers (Turdoides bicolor). ‘Fast-rate chuck’ calls are often given by sentinels as they finish guard bouts and may potentially coordinate the rotation of individuals as sentinels, minimising time without a sentinel, or may signal the presence or absence of predators, regulating the onset of the subsequent sentinel bout. We ask (i) when fast-rate chuck calls are given and (ii) what effect they have on the interval between sentinel bouts. Contrary to expectation, we find little evidence that these calls are involved in regulating the pied babbler sentinel system: observations revealed that their utterance is influenced only marginally by wind conditions and not at all by habitat, while observations and experimental playback showed that the giving of these calls has no effect on inter-bout interval. We conclude that pied babblers do not seem to call at the end of a sentinel bout to maximise the efficiency of this cooperative act, but may use vocalisations at this stage to influence more individually driven behaviours

Mindfulness-based cognitive therapy (MBCT) reduces the association between depressive symptoms and suicidal cognitions in patients with a history of suicidal depression.

Objective: In patients with a history of suicidal depression, recurrence of depressive symptoms can easily reactivate suicidal thinking. In this study, we investigated whether training in mindfulness, which is aimed at helping patients “decenter” from negative thinking, could help weaken the link between depressive symptoms and suicidal cognitions. Method: Analyses were based on data from a recent randomized controlled trial, in which previously suicidal patients were allocated to mindfulness-based cognitive therapy (MBCT), an active control treatment, cognitive psychoeducation (CPE), which did not include any meditation practice, or treatment as usual (TAU). After the end of the treatment phase, we compared the associations between depressive symptoms, as assessed through self-reports on the Beck Depression Inventory–II (Beck, Steer, & Brown, 1996), and suicidal thinking, as assessed through the Suicidal Cognitions Scale (Rudd et al., 2001). Results: In patients with minimal to moderate symptoms at the time of assessment, comparisons of the correlations between depressive symptoms and suicidal cognitions showed significant differences between the groups. Although suicidal cognitions were significantly related to levels of symptoms in the 2 control groups, there was no such relation in the MBCT group. Conclusion: The findings suggest that, in patients with a history of suicidal depression, training in mindfulness can help to weaken the association between depressive symptoms and suicidal thinking, and thus reduce an important vulnerability for relapse to suicidal depression

REC, Drosophila MCM8, Drives Formation of Meiotic Crossovers

Crossovers ensure the accurate segregation of homologous chromosomes from one another during meiosis. Here, we describe the identity and function of the Drosophila melanogaster gene recombination defective (rec), which is required for most meiotic crossing over. We show that rec encodes a member of the mini-chromosome maintenance (MCM) protein family. Six MCM proteins (MCM2–7) are essential for DNA replication and are found in all eukaryotes. REC is the Drosophila ortholog of the recently identified seventh member of this family, MCM8. Our phylogenetic analysis reveals the existence of yet another family member, MCM9, and shows that MCM8 and MCM9 arose early in eukaryotic evolution, though one or both have been lost in multiple eukaryotic lineages. Drosophila has lost MCM9 but retained MCM8, represented by REC. We used genetic and molecular methods to study the function of REC in meiotic recombination. Epistasis experiments suggest that REC acts after the Rad51 ortholog SPN-A but before the endonuclease MEI-9. Although crossovers are reduced by 95% in rec mutants, the frequency of noncrossover gene conversion is significantly increased. Interestingly, gene conversion tracts in rec mutants are about half the length of tracts in wild-type flies. To account for these phenotypes, we propose that REC facilitates repair synthesis during meiotic recombination. In the absence of REC, synthesis does not proceed far enough to allow formation of an intermediate that can give rise to crossovers, and recombination proceeds via synthesis-dependent strand annealing to generate only noncrossover products

A Fully Integrated Real-Time Detection, Diagnosis, and Control of Community Diarrheal Disease Clusters and Outbreaks (the INTEGRATE Project):Protocol for an Enhanced Surveillance System

BACKGROUND:Diarrheal disease, which affects 1 in 4 people in the United Kingdom annually, is the most common cause of outbreaks in community and health care settings. Traditional surveillance methods tend to detect point-source outbreaks of diarrhea and vomiting; they are less effective at identifying low-level and intermittent food supply contamination. Furthermore, it can take up to 9 weeks for infections to be confirmed, reducing slow-burn outbreak recognition, potentially impacting hundreds or thousands of people over wide geographical areas. There is a need to address fundamental problems in traditional diarrheal disease surveillance because of underreporting and subsequent unconfirmed infection by patients and general practitioners (GPs); varying submission practices and selective testing of samples in laboratories; limitations in traditional microbiological diagnostics, meaning that the timeliness of sample testing and etiology of most cases remains unknown; and poorly integrated human and animal surveillance systems, meaning that identification of zoonoses is delayed or missed. OBJECTIVE:This study aims to detect anomalous patterns in the incidence of gastrointestinal disease in the (human) community; to target sampling; to test traditional diagnostic methods against rapid, modern, and sensitive molecular and genomic microbiology methods that identify and characterize responsible pathogens rapidly and more completely; and to determine the cost-effectiveness of rapid, modern, sensitive molecular and genomic microbiology methods. METHODS:Syndromic surveillance will be used to aid identification of anomalous patterns in microbiological events based on temporal associations, demographic similarities among patients and animals, and changes in trends in acute gastroenteritis cases using a point process statistical model. Stool samples will be obtained from patients' consulting GPs, to improve the timeliness of cluster detection and characterize the pathogens responsible, allowing health protection professionals to investigate and control outbreaks quickly, limiting their size and impact. The cost-effectiveness of the proposed system will be examined using formal cost-utility analysis to inform decisions on national implementation. RESULTS:The project commenced on April 1, 2013. Favorable approval was obtained from the Research Ethics Committee on June 15, 2015, and the first patient was recruited on October 13, 2015, with 1407 patients recruited and samples processed using traditional laboratory techniques as of March 2017. CONCLUSIONS:The overall aim of this study is to create a new One Health paradigm for detecting and investigating diarrhea and vomiting in the community in near-real time, shifting from passive human surveillance and management of laboratory-confirmed infection toward an integrated, interdisciplinary enhanced surveillance system including management of people with symptoms. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID):DERR1-10.2196/13941

Common variation in the miR-659 binding-site of GRN is a major risk factor for TDP43-positive frontotemporal dementia

Loss-of-function mutations in progranulin (GRN) cause ubiquitin- and TAR DNA-binding protein 43 (TDP-43)-positive frontotemporal dementia (FTLD-U), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Here we expand the role of GRN in FTLD-U and demonstrate that a common genetic variant (rs5848), located in the 3′-untranslated region (UTR) of GRN in a binding-site for miR-659, is a major susceptibility factor for FTLD-U. In a series of pathologically confirmed FTLD-U patients without GRN mutations, we show that carriers homozygous for the T-allele of rs5848 have a 3.2-fold increased risk to develop FTLD-U compared with homozygous C-allele carriers (95% CI: 1.50–6.73). We further demonstrate that miR-659 can regulate GRN expression in vitro, with miR-659 binding more efficiently to the high risk T-allele of rs5848 resulting in augmented translational inhibition of GRN. A significant reduction in GRN protein was observed in homozygous T-allele carriers in vivo, through biochemical and immunohistochemical methods, mimicking the effect of heterozygous loss-of-function GRN mutations. In support of these findings, the neuropathology of homozygous rs5848 T-allele carriers frequently resembled the pathological FTLD-U subtype of GRN mutation carriers. We suggest that the expression of GRN is regulated by miRNAs and that common genetic variability in a miRNA binding-site can significantly increase the risk for FTLD-U. Translational regulation by miRNAs may represent a common mechanism underlying complex neurodegenerative disorders