Mutational signatures of non‐homologous and polymerase theta‐mediated end‐joining in embryonic stem cells

Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non‐homologous end‐joining (cNHEJ) is largely error‐free, alternative end‐joining pathways have been described that are intrinsically mutagenic. Which end‐joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9‐induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta‐mediated end‐joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end‐joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double‐strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ‐dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.     

Acute myocardial infarction complicating subarachnoid haemorrhage

An acute myocardial infarction is a rare complication of a subarachnoid haemorrhage. The combination of these two conditions imposes important treatment dilemmas. We describe two patients with this combination of life-threatening conditions. Patient 1 was treated with emergency percutaneous coronary intervention followed by clipping of the anterior communicating artery aneurysm. Six months after discharge the patient's memory and orientation had almost completely recovered. Patient 2 was treated with aspirin until coiling of the aneurysm could be performed. After successful coiling low-molecular-weight heparin was added. One week later the patient died due to a free wall rupture. (Neth Heart J 2009;17:284–7.)     

Coral reef recovery dynamics in a changing world

Coral reef ecosystems are degrading through multiple disturbances that are becoming more frequent and severe. The complexities of this degradation have been studied in detail, but little work has assessed characteristics that allow reefs to bounce back and recover between pulse disturbance events. We quantitatively review recovery rates of coral cover from pulse disturbance events among 48 different reef locations, testing the relative roles of disturbance characteristics, reef characteristics, connectivity and anthropogenic influences. Reefs in the western Pacific Ocean had the fastest recovery, whereas reefs in the geographically isolated eastern Pacific Ocean were slowest to recover, reflecting regional differences in coral composition, fish functional diversity and geographic isolation. Disturbances that opened up large areas of benthic space recovered quickly, potentially because of nonlinear recovery where recruitment rates were high. The type of disturbance had a limited effect on subsequent rates of reef recovery, although recovery was faster following crown-of-thorns starfish outbreaks. This inconsequential role of disturbance type may be in part due to the role of unaltered structural complexity in maintaining key reef processes, such as recruitment and herbivory. Few studies explicitly recorded potential ecological determinants of recovery, such as recruitment rates, structural complexity of habitat and the functional composition of reef-associated fish. There was some evidence of slower recovery rates within protected areas compared with other management systems and fished areas, which may reflect the higher initial coral cover in protected areas rather than reflecting a management effect. A better understanding of the driving role of processes, structural complexity and diversity on recovery may enable more appropriate management actions that support coral-dominated ecosystems in our changing climate.     

Kinetics and binding of the thymine-DNA mismatch glycosylase,Mig-Mth,with mismatch-containing DNA substrates

We have examined the removal of thymine residues from T-G mismatches in DNA by the thymine-DNA mismatch glycosylase from Methanobacterium thermoautrophicum (Mig-Mth), within the context of the base excision repair (BER) pathway, to investigate why this glycosylase has such low activity in vitro. Using single-turnover kinetics and steady-state kinetics, we calculated the catalytic and product dissociation rate constants for Mig-Mth, and determined that Mig-Mth is inhibited by product apyrimidinic (AP) sites in DNA. Electrophoretic mobility shift assays (EMSA) provide evidence that the specificity of product binding is dependent upon the base opposite the AP site. The binding of Mig-Mth to DNA containing the non-cleavable substrate analogue difluorotoluene (F) was also analyzed to determine the effect of the opposite base on Mig-Mth binding specificity for substrate-like duplex DNA. The results of these experiments support the idea that opposite strand interactions play roles in determining substrate specificity. Endonuclease IV, which cleaves AP sites in the next step of the BER pathway, was used to analyze the effect of product removal on the overall rate of thymine hydrolysis by Mig-Mth. Our results support the hypothesis that endonuclease IV increases the apparent activity of Mig-Mth significantly under steady-state conditions by preventing reassociation of enzyme to product.     

Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice

Introduction

Biological drugs are effective in patients with rheumatoid arthritis (RA), but increase severe infections. The CC chemokine receptor (CCR) 9 antagonist was effective for Crohn’s disease without critical adverse effects including infections in clinical trials. The present study was carried out to explore the pathogenic roles of chemokine (C-C motif) ligand (CCL) 25 and its receptor, CCR9, in autoimmune arthritis and to study if the CCR9 antagonist could be a new treatment for RA.

Methods

CCL25 and CCR9 expression was examined with immunohistochemistry and Western blotting. Concentration of interleukin (IL)-6, matrix metalloproteinase (MMP)-3 and tumor necrosis factor (TNF)-α was measured with enzyme-linked immunosorbent assays. Effects of abrogating CCR9 on collagen-induced arthritis (CIA) was evaluated using CCR9-deficient mice or the CCR9 antagonist, CCX8037. Fluorescence labeled-CD11b⁺ splenocytes from CIA mice were transferred to recipient CIA mice and those infiltrating into the synovial tissues of the recipient mice were counted.

Results

CCL25 and CCR9 proteins were found in the RA synovial tissues. CCR9 was expressed on macrophages, fibroblast-like synoviocytes (FLS) and dendritic cells in the synovial tissues. Stimulation with CCL25 increased IL-6 and MMP-3 production from RA FLS, and IL-6 and TNF-α production from peripheral blood monocytes. CIA was suppressed in CCR9-deficient mice. CCX8037 also inhibited CIA and the migration of transferred CD11b⁺ splenocytes into the synovial tissues.

Conclusions

The interaction between CCL25 and CCR9 may play important roles in cell infiltration into the RA synovial tissues and inflammatory mediator production. Blocking CCL25 or CCR9 may represent a novel safe therapy for RA.     

DNA Barcoding Reveals Limited Accuracy of Identifications Based on Folk Taxonomy

Background

The trade of plant roots as traditional medicine is an important source of income for many people around the world. Destructive harvesting practices threaten the existence of some plant species. Harvesters of medicinal roots identify the collected species according to their own folk taxonomies, but once the dried or powdered roots enter the chain of commercialization, accurate identification becomes more challenging.

Methodology

A survey of morphological diversity among four root products traded in the medina of Marrakech was conducted. Fifty-one root samples were selected for molecular identification using DNA barcoding using three markers, trnH-psbA, rpoC1, and ITS. Sequences were searched using BLAST against a tailored reference database of Moroccan medicinal plants and their closest relatives submitted to NCBI GenBank.

Principal Findings

Combining psbA-trnH, rpoC1, and ITS allowed the majority of the market samples to be identified to species level. Few of the species level barcoding identifications matched the scientific names given in the literature, including the most authoritative and widely cited pharmacopeia.

Conclusions/Significance

The four root complexes selected from the medicinal plant products traded in Marrakech all comprise more than one species, but not those previously asserted. The findings have major implications for the monitoring of trade in endangered plant species as morphology-based species identifications alone may not be accurate. As a result, trade in certain species may be overestimated, whereas the commercialization of other species may not be recorded at all.     

Bryozoaires des milieux récifaux miocèes du sillon sud-rifain au Maroc

Moissette, P. & Saint Martin, J_:P. 1995 11 30 Bryozoaires des milieux récifaux miocenes du sillon sud-rifain au Maroc. [Bryozoans from Miocene reef environments in the South-Rifian corridor of Morocco. In late Miocene coral reefs from the Fes area (Northern Morocco), bryozoans are the best-represented group. An inventory of the bryozoan fauna (59 species) was taken on two reefs with different organization and palaeogeographical situation in a late Miocene seaway between the Atlantic and the Mediterranean. A study of the bryozoans and their zoarial forms allows an improved reconstruction of reefal environments and available habitats. Membraniporiforrn (encrusting) colonies largely predominate, whereas celleporiforms (nodular colonies) are more rare. Rigid erect (vinculariiform, adeoniform, and reteporiform) and rigid articulated zoarial types (cellariiform and catenicelliform) are fairly well represented. At depths of about 10 m and on the reef front encrusting bryozoans were well developed in cryptic habitats on living coral substrates. Erect species lived in cavities provided by the coral framework, and plants offered flexible substrates for the attachment of epiphytal forms. □Bryozoa, coral reefs, Morocco, Messininn, pulueoecology. Dans les récifs coralliens du Miocène supérieur de la région de Fes (Maroc septentrional), les bryozoaires constituent, parrni les organismes conservés, le groupe le mieux représenté. Un recensement de la fame de bryozoaires (59 espèces) a été effectué dans deux récifs d'organisation et de situation paléogéographique différentes, dans un secteur de communication entre Atlantique et Méditerranée au MiocéFne supérieur. L'étude des bryozoaires et de leurs formes zoariales permet de proposer une meilleure reconstitution de I'environnement récifal et des habitats disponibles. Les colonies membraniporiformes (encrobtantes) predominent largement, alors que les cellepori-formes (colonies noduleuses) sont beaucoup plus rares. Les types zoariaux érigés rigides (vincula-riiforme, adéoniforme et rétéporiforme) et érigés articulés (cellariiforme et catenicelliforme) sont assez bien représentés. A des profondeurs denviron 10 m et dans la partie antérieure des constructions récifales, les bryozoaires encroûtants étaient bien développés dans des habitats cryptiques sur substrats coralliens vivants. Des formes dressées vivaient dans les cavités ménagées par le bâti corallien et des supports végétaux permettaient également la fixation de formes épiphytes. □Bryozouires, récifs coralliens, Muroc, Messinien, puléoécologie.