Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81?CMms4/EME1, Slx1?CSlx4/BTBD12/MUS312, XPF?CERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.     

Synthetic Lethality between Gene Defects Affecting a Single Non-essential Molecular Pathway with Reversible Steps

Systematic analysis of synthetic lethality (SL) constitutes a critical tool for systems biology to decipher molecular pathways. The most accepted mechanistic explanation of SL is that the two genes function in parallel, mutually compensatory pathways, known as between-pathway SL. However, recent genome-wide analyses in yeast identified a significant number of within-pathway negative genetic interactions. The molecular mechanisms leading to within-pathway SL are not fully understood. Here, we propose a novel mechanism leading to within-pathway SL involving two genes functioning in a single non-essential pathway. This type of SL termed within-reversible-pathway SL involves reversible pathway steps, catalyzed by different enzymes in the forward and backward directions, and kinetic trapping of a potentially toxic intermediate. Experimental data with recombinational DNA repair genes validate the concept. Mathematical modeling recapitulates the possibility of kinetic trapping and revealed the potential contributions of synthetic, dosage-lethal interactions in such a genetic system as well as the possibility of within-pathway positive masking interactions. Analysis of yeast gene interaction and pathway data suggests broad applicability of this novel concept. These observations extend the canonical interpretation of synthetic-lethal or synthetic-sick interactions with direct implications to reconstruct molecular pathways and improve therapeutic approaches to diseases such as cancer.     

Microbiota and SCFA in Lean and Overweight Healthy Subjects

Obesity has recently been linked to the composition of human microbiota and the production of short chain fatty acids (SCFAs). However, these findings rely on experimental studies carried out using rather small and defined groups of volunteers or model animals. Our aim was to evaluate differences within the human intestinal microbiota and fecal SCFA concentration of lean and obese subjects. A total of 98 subjects volunteered to take part in this study. The BMI in kg/m² of 30 volunteers was within the lean range, 35 were overweight and 33 were obese. The fecal microbiota was characterized by real‐time PCR analyses. With the primers used herein we were able to cover 82.3% (interquartile range of 68.3–91.4%) of the total microbiota detectable with a universal primer. In addition, the concentration of SCFA was evaluated. The total amount of SCFA was higher in the obese subject group (P = 0.024) than in the lean subject group. The proportion of individual SCFA changed in favor of propionate in overweight (P = 0.019) and obese subjects (P = 0.028). The most abundant bacterial groups in faeces of lean and obese subjects belonged to the phyla Firmicutes and Bacteroidetes. The ratio of Firmicutes to Bacteroidetes changed in favor of the Bacteroidetes in overweight (P = 0.001) and obese subjects (P = 0.005). Our results are in line with previous reports suggesting that SCFA metabolism might play a considerable role in obesity. However, our results contradict previous reports with regard to the contribution of various bacterial groups to the development of obesity and this issue remains controversial.     

New Aspects on the Structure of Neutrophil Extracellular Traps from Chronic Obstructive Pulmonary Disease and In Vitro Generation

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in ‘beads-on-a-string’ conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of ‘beads-on-a-string’ DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.     

Wie aus Zwergen Riesen wurden. Eine kurze Geschichte der Dinosaurier Argentiniens

Argentinian dinosaurs Dinosaurs are amazing! We enjoy imagining how they lived, walked around, what they ate and the way they fight. First of all the Giants attract our interest – the long‐necked herbivore sauropods and the dreadful predatory dinosaurs. Argentina offers fantastic conditions concerning the reconstruction of the successful dinosaur evolution and Gigantism respectively. Fossils of the “terrible lizards” are found through all the three eras of the Mesozoic – Triassic, Jurassic and Cretaceous. A potential forerunner of the dinosaurs – an archosaur – was the size of a hen. Eoraptor, the oldest known real dinosaur, was only as large as a little dog. In contrast, Argentinosaurus huinculensis was a 40 Meter long sauropod weighting 80 tons.     

Salmonella-induced mucosal lectin RegIIIβ kills competing gut microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.     

Efficient Generation of Rat Induced Pluripotent Stem Cells Using a Non-Viral Inducible Vector

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.     

Vergesellschaftung als Verhaltensbereicherung im Zoo – Soziale Interaktion und Raumnutzung bei Orang-Utans,Hulmans und Kurzkrallenottern in der Zoom Erlebniswelt Gelsenkirchen

Keeping different species together is getting more and more common in zoos, because it can entail several behavioural enrichment factors for the animals as long as the enclosure meets the requirements of all species and a suitable composition of animals is living in the mix. The aim of this study is to evaluate a community of Sumatran orangutans (Pongo abelii), northern plains grey langurs (Semnopithecus entellus) and Asian small-clawed otters (Aonyx cinerea) in the Zoological Garden of Gelsenkirchen.The behavioural monitoring of the animals reveals that the orangutans spend less time with feeding behaviour and locomotion than free ranging orangutans, whereas the percentage of resting periods is much higher. This difference is probably caused by the easy availability of food. The northern plains grey langurs show ethograms whose proportions are very similar to those of free ranging grey langurs in literature. In both species the juvenile individuals have higher activity levels than the adult animals.The analysis of the spatial use of the enclosure by the orangutans reveals that they use about half of the area and prefer places at the ground for resting. On an average the orangutans spend around 50% of the monitored situations on the ground which is a high level for an arboreal living species. The time spent on the ground is distinctly lower in orangutans reared by their parents than in hand reared individuals which are probably affected by humans. Another influencing point is the sex. Adult male orangutans spend more time on the ground. This is a fact which is also known from free ranging animals. Related to their environment free ranging northern plains grey langurs show a high adaptability and the individuals in the zoo use about 70% of the enclosure and use frequently all structuring elements like trees, roots and ropes. Like the orangutans the grey langurs have preferred areas in the enclosure where they spend their resting periods with grooming, lactating and the feeding periods. Generally, the Asian small-clawed otters spend less time in the enclosure than the monkeys and use less than one third of the area.Most of the time free ranging orangutans live solitarily and thus it was predictable that there is only a fraction of intraspecific interactions between orangutans whereas the percentage of interactions between langurs is higher. The northern plains grey langurs live in harem troops and social interactions are more than 40% of the zoo langur's ethogram on an average. Asian small-clawed otters are also highly social animals and intraspecific contacts take place very often.The analysis of the monitored interspecific interactions between all species reveals that the juvenile animals of both primate species have distinct more interspecific contacts than the adult animals. In most cases the interactions are positive or playful contacts. Especially between a 12-year-old female orang-utan and one of the juvenile langurs friendly interactions with body contact were monitored often. Between the adult animals agonistic contacts or submission are most common. The Asian small-clawed otters are very interested in the orangutans and often approach towards them to have a sniff or pull their hair.The community of all three species is quite harmonious and serves as behavioural enrichment for all individuals. For the visitors the mixed enclosure is a benefit as well.