REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Aortic morphometry at endograft position as assessed by 3D image analysis affects risk of type I endoleak formation after TEVAR

PURPOSE The purpose of this study was to identify morphologic factors affecting type I endoleak formation and bird-beak configuration after thoracic endovascular aortic repair (TEVAR). METHODS Computed tomography (CT) data of 57 patients (40 males; median age, 66 years) undergoing TEVAR for thoracic aortic aneurysm (34 TAA, 19 TAAA) or penetrating aortic ulcer (n = 4) between 2001 and 2010 were retrospectively reviewed. In 28 patients, the Gore TAG® stent-graft was used, followed by the Medtronic Valiant® in 16 cases, the Medtronic Talent® in 8, and the Cook Zenith® in 5 cases. Proximal landing zone (PLZ) was in zone 1 in 13, zone 2 in 13, zone 3 in 23, and zone 4 in 8 patients. In 14 patients (25%), the procedure was urgent or emergent. In each case, pre- and postoperative CT angiography was analyzed using a dedicated image processing workstation and complimentary in-house developed software based on a 3D cylindrical intensity model to calculate aortic arch angulation and conicity of the landing zones (LZ). RESULTS Primary type Ia endoleak rate was 12% (7/57) and subsequent re-intervention rate was 86% (6/7). Left subclavian artery (LSA) coverage (p = 0.036) and conicity of the PLZ (5.9 vs. 2.6 mm; p = 0.016) were significantly associated with an increased type Ia endoleak rate. Bird-beak configuration was observed in 16 patients (28%) and was associated with a smaller radius of the aortic arch curvature (42 vs. 65 mm; p = 0.049). Type Ia endoleak was not associated with a bird-beak configuration (p = 0.388). Primary type Ib endoleak rate was 7% (4/57) and subsequent re-intervention rate was 100%. Conicity of the distal LZ was associated with an increased type Ib endoleak rate (8.3 vs. 2.6 mm; p = 0.038). CONCLUSIONS CT-based 3D aortic morphometry helps to identify risk factors of type I endoleak formation and bird-beak configuration during TEVAR. These factors were LSA coverage and conicity within the landing zones for type I endoleak formation and steep aortic angulation for bird-beak configuration.

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make them incompatible with testing in the resource-poor settings that are most relevant to this disease. We report a rapid, specific, and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.

Measurement of fecal elastase improves performance of newborn screening for cystic fibrosis.

BACKGROUND The aim of newborn screening (NBS) for CF is to detect children with 'classic' CF where early treatment is possible and improves prognosis. Children with inconclusive CF diagnosis (CFSPID) should not be detected, as there is no evidence for improvement through early treatment. No algorithm in current NBS guidelines explains what to do when sweat test (ST) fails. This study compares the performance of three different algorithms for further diagnostic evaluations when first ST is unsuccessful, regarding the numbers of children detected with CF and CFSPID, and the time until a definite diagnosis. METHODS In Switzerland, CF-NBS was introduced in January 2011 using an IRT-DNA-IRT algorithm followed by a ST. In children, in whom ST was not possible (no or insufficient sweat), 3 different protocols were applied between 2011 and 2014: in 2011, ST was repeated until it was successful (protocol A), in 2012 we proceeded directly to diagnostic DNA testing (protocol B), and 2013-2014, fecal elastase (FE) was measured in the stool, in order to determine a pancreas insufficiency needing immediate treatment (protocol C). RESULTS The ratio CF:CFSPID was 7:1 (27/4) with protocol A, 2:1 (22/10) with protocol B, and 14:1 (54/4) with protocol C. The mean time to definite diagnosis was significantly shorter with protocol C (33days) compared to protocol A or B (42 and 40days; p=0.014 compared to A, and p=0.036 compared to B). CONCLUSIONS The algorithm for the diagnostic part of the newborn screening used in the CF centers is important and affects the performance of a CF-NBS program with regard to the ratio CF:CFSPID and the time until definite diagnosis. Our results suggest to include FE after initial sweat test failure in the CF-NBS guidelines to keep the proportion of CFSPID low and the time until definite diagnosis short.

IGHD II: A Novel GH-1 Gene Mutation (GH-L76P) Severely Affects GH Folding, Stability, and Secretion

CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.

Cognitively engaging chronic physical activity, but not aerobic exercise, affects executive functions in primary school children: A group-randomized controlled trial

Although the positive effects of different kinds of physical activity (PA) on cognitive functioning have already been demonstrated in a variety of studies, the role of cognitive engagement in promoting children’s executive functions is still unclear. The aim of the present study was therefore to investigate the effects of two qualitatively different chronic PA interventions on executive functions in primary school children. 181 children aged between 10 and 12 years were assigned to either a 6-week physical education program with a high level of physical exertion and high cognitive engagement (team games), a physical education program with high physical exertion but low cognitive engagement (aerobic exercise), or to a physical education program with both low physical exertion and low cognitive engagement (control condition). Executive functions (updating, inhibition, shifting) and aerobic fitness (multistage 20-meter shuttle run test) were measured before and after the respective condition. Results revealed that both interventions (team games and aerobic exercise) have a positive impact on children’s aerobic fitness (4-5 % increase in estimated VO2max). Importantly, an improvement in shifting performance was found only in the team games and not in the aerobic exercise or control condition. Thus, the inclusion of cognitive engagement in PA seems to be the most promising type of chronic intervention to enhance executive functions in children, providing further evidence for the importance of the qualitative aspects of PA.

Doxorubicin Affects Expression of Proteins of Neuronal Pathways in MCF-7 Breast Cancer Cells.

In the present article, we report on the semi-quantitative proteome analysis and related changes in protein expression of the MCF-7 breast cancer cell line following treatment with doxorubicin, using the precursor acquisition independent from ion count (PAcIFIC) mass spectrometry method. PAcIFIC represents a cost-effective and easy-to-use proteomics approach, enabling for deep proteome sequencing with minimal sample handling. The acquired proteomic data sets were searched for regulated Reactome pathways and Gene Ontology annotation terms using a new algorithm (SetRank). Using this approach, we identified pathways with significant changes (≤0.05), such as chromatin organization, DNA binding, embryo development, condensed chromosome, sequence-specific DNA binding, response to oxidative stress and response to toxin, as well as others. These sets of pathways are already well-described as being susceptible to chemotherapeutic drugs. Additionally, we found pathways related to neuron development, such as central nervous system neuron differentiation, neuron projection membrane and SNAP receptor activity. These later pathways might indicate biological mechanisms on the molecular level causing the known side-effect of doxorubicin chemotherapy, characterized as cognitive impairment, also called 'chemo brain'. Mass spectrometry data are available via ProteomeXchange with identifier PXD002998.

Plant genotype affects the quality of oilseed rape (Brassica napus) for adults and larvae of the pollen beetle (Meligethes aeneus)

Plant quality is one of the main factors influencing the fitness of phytophagous insects. Plant quality can vary not only among genotypes of the same host plant species, but also relative to the insect sex or its life stage. In the present study, the performance of larvae and adults of the pollen beetle (Meligethes aeneus F., Coleoptera: Nitidulidae), a major insect pest of oilseed rape crops, is compared on six genotypes of oilseed rape (Brassica napus). All of the traits that are measured vary among genotypes, and comprise larval developmental duration, life span of unfed emerging adults and survival time of field-sampled adults fed with pollen from the different genotypes. No correlation is found between insect performance and quantity of food available, showing that the quality of the food (i.e. pollen) is the fitness determinant for this insect species. Additionally, the performance of larvae and adults is also not correlated despite use of the same plant genotypes, suggesting that the determinants of pollen quality differ at least partially between both life stages. It is hypothesized that this may be a result of extensive differences in diet breadth between the life stages: larvae are specialists of brassicaceous plants, whereas adults are generalists. Finally, it is suggested that the manipulation of plant quality to increase pollen beetle development time may comprise a valuable strategy for favouring biological control by natural enemies of this pest; for example, as a result of extending the vulnerability window of larvae to attack by parasitoids.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti-MHC class II Abs is not due to interference with the state of activation of the T cells since the transcriptional activator NF-kappa B remains activated in arrested cells. In addition, interleukin 2 (IL-2), IL-2R, and c-myc gene expression are also unaffected. By analyzing the cell-cycle phase distribution of inhibited cells, it could be shown that cells in all phases of the cell cycle are inhibited. The signal transduction pathway that results in inhibition was shown to be independent of protein kinase C and extracellular Ca2+. Tyrosine kinase inhibitors, however, partly reduced the level of inhibition and, conversely, phosphatase inhibitors enhanced it. The possible relevance of this phenomenon in other systems is discussed.

Are fractured cliffs the source of cometary dust jets? Insights from OSIRIS/Rosetta at 67P/Churyumov-Gerasimenko

Context. Dust jets (i.e., fuzzy collimated streams of cometary material arising from the nucleus) have been observed in situ on all comets since the Giotto mission flew by comet 1P/Halley in 1986, and yet their formation mechanism remains unknown. Several solutions have been proposed involving either specific properties of the active areas or the local topography to create and focus the gas and dust flows. While the nucleus morphology seems to be responsible for the larger features, high resolution imagery has shown that broad streams are composed of many smaller jets (a few meters wide) that connect directly to the nucleus surface. Aims. We monitored these jets at high resolution and over several months to understand what the physical processes are that drive their formation and how this affects the surface. Methods. Using many images of the same areas with different viewing angles, we performed a 3-dimensional reconstruction of collimated jets and linked them precisely to their sources on the nucleus. Results. We show here observational evidence that the northern hemisphere jets of comet 67P/Churyumov-Gerasimenko arise from areas with sharp topographic changes and describe the physical processes involved. We propose a model in which active cliffs are the main source of jet-like features and therefore of the regions eroding the fastest on comets. We suggest that this is a common mechanism taking place on all comets.

Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to “stress”: pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2–3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.

Genetic selection to increase bone strength affects prevalence of keel bone damage and egg parameters in commercially housed laying hens.

The prevalence of keel bone damage as well as external egg parameters of 2 pure lines divergently selected for high (H) and low (L) bone strength were investigated in 2 aviary systems under commercial conditions. A standard LSL hybrid was used as a reference group. Birds were kept mixed per genetic line (77 hens of the H and L line and 201 or 206 hens of the LSL line, respectively, per pen) in 8 pens of 2 aviary systems differing in design. Keel bone status and body mass of 20 focal hens per line and pen were assessed at 17, 18, 23, 30, 36, 43, 52, and 63 wk of age. External egg parameters (i.e., egg mass, eggshell breaking strength, thickness, and mass) were measured using 10 eggs per line at both 38 and 57 wk of age. Body parameters (i.e. tarsus and third primary wing feather length to calculate index of wing loading) were recorded at 38 wk of age and mortality per genetic line throughout the laying cycle. Bone mineral density (BMD) of 15 keel bones per genetic line was measured after slaughter to confirm assignment of the experimental lines. We found a greater BMD in the H compared with the L and LSL lines. Fewer keel bone fractures and deviations, a poorer external egg quality, as well as a lower index of wing loading were found in the H compared with the L line. Mortality was lower and production parameters (e.g., laying performance) were higher in the LSL line compared with the 2 experimental lines. Aviary design affected prevalence of keel bone damage, body mass, and mortality. We conclude that selection of specific bone traits associated with bone strength as well as the related differences in body morphology (i.e., lower index of wing loading) have potential to reduce keel bone damage in commercial settings. Also, the housing environment (i.e., aviary design) may have additive effects.