Checkpoints: Chromosome pairing takes an unexpected twist

When meiotic cells complete S phase, homologous chromosomes pair, synapse and undergo recombination. A checkpoint protein is somehow required for meiotic chromosome pairing in C. elegans, thus providing a direct link between S phase and the rest of the meiotic program

Functional Analysis of the Yku Complex in Telomere Length Regulation

The Ku protein from Saccharomyces cerevisiae (Yku) forms, like its human homologue hKu, a heterodimer comprised of a 70kD and an 80 kD subunit. In yeast and mammals, the Ku heterodimer is required for the repair of DNA double strand breaks (DSBs) via nonhomologous end-joining (NHEJ). Interestingly, Ku has been shown to bind to the native chromosome ends. It contributes to the maintenance of wild type telomere length and, moreover, has been implicated in the protection of the telomeres from end-to-end fusions. Telomere-bound Yku delocalizes from telomeric foci in response to DNA damage and accumulates at the sites of a DNA break. This thesis aimed to further characterize the Yku heterodimer and its function at DSBs and the native chromosome ends. In a genetic screen for mutations that - in combination with a yku deletion - lead to cell death, a novel mutation in the yeast telomerase subunit CDC13/EST4 has been identified earlier in the laboratory. Cdc13p binds to the single stranded DNA overhang at telomeres and is required to recruit the yeast telomerase to chromosome ends. The results presented here suggest that the mutant protein, Cdc13-4p, can still bind to the telomere and does interact with the telomerase subunit Est1p in vivo. A model is proposed in which the mutant Cdc13p is altered in its binding to a regulatory protein, thereby modulating telomerase access to the chromosome ends. The lethal effect in yku mutants is discussed to result from the loss of additional telomere sequences at the already very short telomeres of yku mutants. In order to fulfill its opposite functions at the ‘different’ DNA ends, Yku might depend on larger protein networks. Putative Yku interacting proteins have been identified in a two hybrid screen. One interactor, Sir4p, has previously been implicated in NHEJ. The Sir4p domain identified could be shown to interact with the Yku heterodimer via the Yku80p subunit. Experiments that allowed the separation of phenotypes caused by the loss of the Sir4 protein itself and phenotypes induced by a de-repression of silencing in sir4 mutants revealed no direct involvement of Sir4p in the repair of DSBs. In contrast to Cdc13p, Sir4p acts epistatic with Yku at the telomeres, indicating that the protein-protein interaction detected by two hybrid criteria might take place at telomeres. Besides defects in DNA repair and telomere protection, mice deficient for Ku have been reported to exhibit phenotypes indicative of premature aging. Loss of yku70 or overexpression of the Yku heterodimer effects life span in yeast. Experiments presented here rise the possibility that the premature aging is correlated with Ku’s function at the telomere

Impact of clopidogrel in coronary artery bypass grafting

Objective: Clopidogrel has become the standard of care to prevent thrombotic complications following cardiological interventions, in particular intracoronary stenting. In addition, patients with aspirin intolerance and those with carotid and peripheral vascular disease are also increasingly treated with clopidogrel. Platelet inhibition may become a concern for hemostasis in patients treated with clopidogrel who need emergency and undelayed surgery. Methods: We prospectively analyzed the intra- and postoperative outcome of 505 consecutive patients who underwent isolated CABG and compared two groups: those with clopidogrel exposure until 72 h prior to surgery (n=136) with those without exposition to clopidogrel (n=369). Patients undergoing emergency surgery because of failed PTCA and cardiogenic shock, associated valvular surgery, redo-CABG, and those with additional platelet IIb/IIIa receptor inhibitor exposure were excluded. Patients who received aspirin and/or heparin treatment prior to surgery were not excluded. Results: Patients who received clopidogrel had a higher prevalence of angina class III or IV (67 vs 39%, P<0.01), received more often revascularization within 48 h (41 vs 14%, P=0.02), and had received more frequently stenting (57 vs 13%). Chest tube drainage was significantly increased during the first 24 h following CABG in the group of patients who had clopidogrel treatment (1485 vs 780 ml, P=0.003) These patients also required more transfusion of platelets and fresh frozen plasma. Overall re-exploration rate because of bleeding was significantly higher in the clopidogrel group (5.9 vs 1.2%, P<0.01). Platelets transfused before chest closure had a beneficial effect on preservation of the hemostasis. Conclusions: Clopidogrel exposure 3 days or less prior to CABG surgery significantly increases the risk of postoperative bleeding, the need for perioperative transfusion and the incidence of re-exploration. Surgery should be performed using standard heparinization and anti-fibrinolytic strategies but aggressive correction of platelets dysfunction is required before chest closur

Significance of Coprophagy for the Fatty Acid Profile in Body Tissues of Rabbits Fed Different Diets

Four groups of eight New Zealand hybrid rabbits were fattened with ad libitum access to the following pelleted experimental diets: ryegrass meal or alfalfa meal fed either alone or with oats meal in a ratio of 1:1. After 25weeks they were slaughtered and dissected. Fatty acid (FA) profiles of caecotrophs (re-ingested fermentation products of the caecum), perirenal adipose tissue and intramuscular fat in the Musculus quadriceps were determined. With high proportions of branched-chain FA (BFA) and trans FA, and increased proportions of saturated FA relative to the diets, the caecotroph FA profile showed a clear fingerprint of anaerobe microbial lipid metabolism including biohydrogenation. By contrast, the FA profiles of adipose and lean tissue comprised high proportions of polyunsaturated FA (PUFA), whilst BFA and trans FA occurred in much lower proportions compared to the caecotrophs. Thus, coprophagy did not substantially modify the FA composition of the tissues investigated. Use of forage-only diets, compared to the oats supplemented diets, led to extraordinary high proportions of n-3 PUFA (including 18:3 and long-chain n-3) in the fat of adipose (21.3 vs. 6.7%) and lean tissue (15.4 vs. 5.7%). The forage type diet (grass vs. alfalfa) had smaller effects on the FA profiles. Indications of diet effects on endogenous desaturation, chain elongation and differential distribution of functional FA between the two tissues investigated were foun

Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase epsilon subunit pole-4. Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of N (C) under barG &gt; NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes

trt-1 Is the Caenorhabditis elegans Catalytic Subunit of Telomerase

Mutants of trt-1, the Caenorhabditis elegans telomerase reverse transcriptase, reproduce normally for several generations but eventually become sterile as a consequence of telomere erosion and end-to-end chromosome fusions. Telomere erosion and uncapping do not cause an increase in apoptosis in the germlines of trt-1 mutants. Instead, late-generation trt-1 mutants display chromosome segregation defects that are likely to be the direct cause of sterility. trt-1 functions in the same telomere replication pathway as mrt-2, a component of the Rad9/Rad1/Hus1 (9–1–1) proliferating cell nuclear antigen–like sliding clamp. Thus, the 9–1–1 complex may be required for telomerase to act at chromosome ends in C. elegans. Although telomere erosion limits replicative life span in human somatic cells, neither trt-1 nor telomere shortening affects postmitotic aging in C. elegans. These findings illustrate effects of telomere dysfunction in C. elegans mutants lacking the catalytic subunit of telomerase, trt-1

Raufutter als Alleinfutter für Kaninchen – Auswirkungen auf das Fettsäurenmuster des Fleisches

Four groups of eight New Zealand hybrid rabbits were fed ryegrass meal only, alfalfa meal only, ryegrass and oats 1:1, or alfalfa and oats 1:1. Diets were offered ad libitum in pelleted form from 5-30 weeks of age, when they were slaughtered. Intramuscular fatty acid profiles were determined in the Musculus quadriceps of the left hindleg. Feed intake was not statistically different between the four groups, nor was carcass weight. The main effect of the forage-only diets on the fatty acid profiles was a decrease of monounsaturated and an increase of polyunsaturated fatty acid (PUFA) proportions. Within PUFA, the n-3 fatty acids more than doubled with forage-only compared to forage-oats diets, while the n-6 fatty acids slightly decreased. In general, the proportion of n-3 fatty acids in intramuscular fat of forage-only fed rabbits was extraordinarily high compared to any other meat of agricultural origin. The results demonstrate a specific advantage of roughage-based diets in the nutrition of productive herbivores, which is also known for ruminants

Having a direct look:analysis of DNA damage and repair mechanisms by next generation sequencing

AbstractGenetic information is under constant attack from endogenous and exogenous sources, and the use of model organisms has provided important frameworks to understand how genome stability is maintained and how various DNA lesions are repaired. The advance of high throughput next generation sequencing (NGS) provides new inroads for investigating mechanisms needed for genome maintenance. These emerging studies, which aim to link genetic toxicology and mechanistic analyses of DNA repair processes in vivo, rely on defining mutational signatures caused by faulty replication, endogenous DNA damaging metabolites, or exogenously applied genotoxins; the analysis of their nature, their frequency and distribution. In contrast to classical studies, where DNA repair deficiency is assessed by reduced cellular survival, the localization of DNA repair factors and their interdependence as well as limited analysis of single locus reporter assays, NGS based approaches reveal the direct, quantal imprint of mutagenesis genome-wide, at the DNA sequence level. As we will show, such investigations require the analysis of DNA derived from single genotoxin treated cells, or DNA from cell populations regularly passaged through single cell bottlenecks when naturally occurring mutation accumulation is investigated. We will argue that the life cycle of the nematode Caenorhabditis elegans, its genetic malleability combined with whole genome sequencing provides an exciting model system to conduct such analysis

C. elegans genome-wide analysis reveals DNA repair pathways that act cooperatively to preserve genome integrity upon ionizing radiation.

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions

List-method directed forgetting: Do critical findings generalize from short to long retention intervals?

People can purposefully forget information that has become irrelevant, as is demonstrated in list-method directed forgetting (LMDF). In this task, participants are cued to intentionally forget an already studied list (list 1) before encoding a second list (list 2); this induces forgetting of the first-list items. Most research on LMDF has been conducted with short retention intervals, but very recent studies indicate that such directed forgetting can be lasting. We examined in two experiments whether core findings in the LMDF literature generalize from short to long retention intervals. The focus of Experiment 1 was on the previous finding that, with short retention interval, list-2 encoding is necessary for list-1 forgetting to arise. Experiment 1 replicated the finding after a short delay of 3 min between study and test and extended it to a longer delay of 20 min. The focus of Experiment 1 was on the absence of list-1 forgetting in item recognition, previously observed after short retention interval. Experiment 1 replicated the finding after a short delay of 3 min between study and test and extended it to longer delays of 20 min and 24 h. Implications of the results for theoretical explanations of LMDF are discussed