BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells’ (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.Subject terms: Breast cancer, Cancer stem cells     

Non Digestible Oligosaccharides Modulate the Gut Microbiota to Control the Development of Leukemia and Associated Cachexia in Mice

We tested the hypothesis that changing the gut microbiota using pectic oligosaccharides (POS) or inulin (INU) differently modulates the progression of leukemia and related metabolic disorders. Mice were transplanted with Bcr-Abl-transfected proB lymphocytes mimicking leukemia and received either POS or INU in their diet (5%) for 2 weeks. Combination of pyrosequencing, PCR-DGGE and qPCR analyses of the 16S rRNA gene revealed that POS decreased microbial diversity and richness of caecal microbiota whereas it increased Bifidobacterium spp., Roseburia spp. and Bacteroides spp. (affecting specifically B. dorei) to a higher extent than INU. INU supplementation increased the portal SCFA propionate and butyrate, and decreased cancer cell invasion in the liver. POS treatment did not affect hepatic cancer cell invasion, but was more efficient than INU to decrease the metabolic alterations. Indeed, POS better than INU delayed anorexia linked to cancer progression. In addition, POS treatment increased acetate in the caecal content, changed the fatty acid profile inside adipose tissue and counteracted the induction of markers controlling β-oxidation, thereby hampering fat mass loss. Non digestible carbohydrates with prebiotic properties may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.     

Criteria for assessing maturity of skulls in the common dolphin,Delphinus sp., from New Zealand waters

Knowledge about the maturity status of specimens included in evolutionary, taxonomic or life history investigations is fundamentally important. This study investigated the use of the degree of cranial suture fusion, the developmental status of cranial bones, and the degree of tooth wear as indicators for cranial maturity status in Delphinus sp. from New Zealand waters. In total, 15 sutures, one joint and three nonmetric characters were assessed on 66 skulls obtained from stranded and bycaught individuals sampled between 1932 and 2011. A suture index (SI) was computed based on 10 sutures, in which degree of fusion was correlated with age and the three misclassification indices (MI), calculated for a given suture, were <50%. In addition to these, five premaxilla‐maxilla fusion and seven tooth wear categories were assessed. Results suggest that New Zealand Delphinus sp. skulls should be regarded as cranially mature if at least two of the following criteria are met: (1) individuals assessed as sexually mature, (2) aged ≥ 11 yr, (3) SI ≥ 8, and (4) premaxilla‐maxilla fusion ≥ 75% of the length of the dorsal side of the rostrum. Presence of any number of rostral teeth worn to the gum line provided further evidence for cranial maturity.     

Immunogenetic heterogeneity in a widespread ungulate: the European roe deer (Capreolus capreolus)

Understanding how immune genetic variation is shaped by selective and neutral processes in wild populations is of prime importance in both evolutionary biology and epidemiology. The European roe deer (Capreolus capreolus) has considerably expanded its distribution range these last decades, notably by colonizing agricultural landscapes. This range shift is likely to have led to bottlenecks and increased roe deer exposure to a new range of pathogens that until recently predominantly infected humans and domestic fauna. We therefore investigated the historical and contemporary forces that have shaped variability in a panel of genes involved in innate and acquired immunity in roe deer, including Mhc‐Drb and genes encoding cytokines or toll‐like receptors (TLRs). Together, our results suggest that genetic drift is the main contemporary evolutionary force shaping immunogenetic variation within populations. However, in contrast to the classical view, we found that some innate immune genes involved in micropathogen recognition (e.g. Tlrs) continue to evolve dynamically in roe deer in response to pathogen‐mediated positive selection. Most studied Tlrs (Tlr2, Tlr4 and Tlr5) had similarly high levels of amino acid diversity in the three studied populations including one recently established in southwestern France that showed a clear signature of genetic bottleneck. Tlr2 implicated in the recognition of Gram‐positive bacteria in domestic ungulates, showed strong evidence of balancing selection. The high immunogenetic variation revealed here implies that roe deer are able to cope with a wide spectrum of pathogens and to respond rapidly to emerging infectious diseases.     

Detection of Antibiotic Resistance in Leprosy Using GenoType LepraeDR, a Novel Ready-To-Use Molecular Test

Background

Although leprosy is efficiently treated by multidrug therapy, resistance to first-line (dapsone, rifampin) and to second-line drugs (fluoroquinolones) was described worldwide. Since Mycobacterium leprae is not growing in vitro, phenotypic susceptibility testing requires a one year experiment in the mouse model and this is rarely performed. Genetics on antibiotic resistance provide the basis for molecular tests able to detect for antibiotic resistance in leprosy.

Methodology/Principal Findings

A reverse hybridization DNA strip test was developed as the GenoType LepraeDR test. It includes DNA probes for the wild-type sequence of regions of rpoB, gyrA and folP genes and probes for the prevalent mutations involved in acquired resistance to rifampin, fluoroquinolones and dapsone, respectively. The performances of the GenoType LepraeDR test were evaluated by comparing its results on 120 M. leprae strains, previously studied for resistance by the reference drug in vivo susceptibility method in the mouse footpad and for mutations in the gene regions described above by PCR-sequencing. The results of the test were 100% concordant with those of PCR sequencing and the mouse footpad test for the resistant strains: 16 strains resistant to rifampin, 22 to dapsone and 4 to ofloxacin with mutations (numbering system of the M. leprae genome) in rpoB (10 S456L, 1 S456F, 1 S456M + L458V, 1 H451Y, 1 G432S + H451D, 1 T433I + D441Y and 1 Q438V), in folP1 (8 P55L, 3 P55R, 7 T53I, 3 T53A, 1 T53V) and gyrA (4 A91V), respectively. Concordance was 98.3% for the susceptible strains, two strains showing a mutation at the codon 447 that in fact was not conferring resistance as shown by the in vivo method.

Conclusions/Significance

The GenoType LepraeDR test is a commercially available test that accurately detects for antibiotic resistance in leprosy cases. The test is easy to perform and could be implemented in endemic countries.     

Identification of a series of 1,3,4-oxadiazol-2-amines as potent alpha-7 agonists with efficacy in the novel object recognition model of cognition

A series of α7 nicotinic acetylcholine receptor full-agonists with a 1,3,4-oxadiazol-2-amine core has been discovered. Systematic exploration of the structure-activity relationships for both α7 potency and selectivity with respect to interaction with the hERG channel are described. Further profiling led to the identification of compound 22, a potent full agonist showing efficacy in the novel object recognition model of cognition enhancement.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

Proteomic studies highlight outer‐membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer‐membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer‐membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB‐dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT‐PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.     

Application of the Three Rs to challenge assays used in vaccine testing: Tenth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement

This report aims to facilitate the implementation of the Three Rs (reduction, refinement and replacement) in the testing of vaccines for regulatory and other purposes. The focus is predominantly on identification of reduction and refinement opportunities in batch potency testing but the principles described are widely applicable to other situations that involve experimental infections of animals. The report should also help to interpret the requirements of the European Pharmacopoeia with regard to the use of alternative tests, humane endpoints and other refinements. Two specific worked examples, for batch potency testing of Clostridium chauvoei and canine leptospira, with recommendations for harmonisation of international test requirements for these and other vaccines, are provided as appendices online.