Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia

DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM), which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env) of human immunodeficiency virus-1 (HIV-1) in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein), as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.     

Glycine betaine loses its osmoprotective activity in a bspA strain of Erwinia chrysanthemi

Erwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high-salt media. Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase. Initial GB uptake is not affected by the bspA mutation. However, in high-salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability. BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel. We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high-salt media containing this osmoprotectant.     

Prostaglandins antagonistically control Bax activation during apoptosis

The Bax protein (Bcl-2-associated X protein) is pivotal for the apoptotic process. Bax, which resides in an inactive form in the cytosol of healthy cells, is activated during the early stages of apoptosis and becomes associated with mitochondria through poorly understood mechanisms. In this study, we show that a family of bioactive lipids, namely prostaglandins, regulates Bax-dependent apoptosis. The prostaglandin E(2) (PGE(2)) or its derivative PGA(2) binds to Bax, induces its change of conformation, and thereby triggers apoptosis. A cysteine present in the loop between the two transmembrane α-helices of Bax, Cys126 is critical for its activation. PGD(2) inhibits PGE(2) binding to Bax and PGE(2)-induced apoptosis, as well as cell death induced by staurosporine and UV-B in various cell lines. This result is consistent with the fact that apoptosis is accompanied during these treatments by an increase in PGE(2). This process is distinct, yet cooperative, from that involving the BH3-only protein Bid. Our results establish that the PGE(2)/PGD(2) balance is involved in a new early mechanism of control in the activation of Bax during apoptosis.     

Genetic diversity and investigation of polledness in divergent goat populations using 52 088 SNPs

The recent availability of a genome‐wide SNP array for the goat genome dramatically increases the power to investigate aspects of genetic diversity and to conduct genome‐wide association studies in this important domestic species. We collected and analysed genotypes from 52 088 SNPs in Boer, Cashmere and Rangeland goats that had both polled and horned individuals. Principal components analysis revealed a clear genetic division between animals for each population, and model‐based clustering successfully detected evidence of admixture that matched aspects of their recorded history. For example, shared co‐ancestry was detected, suggesting Boer goats have been introgressed into the Rangeland population. Further, allele frequency data successfully tracked the altered genetic profile that has taken place after 40 years of breeding Australian Cashmere goats using the Rangeland animals as the founding population. Genome‐wide association mapping of the POLL locus revealed a strong signal on goat chromosome 1. The 769‐kb critical interval contained the polled intersex syndrome locus, confirming the genetic basis in non‐European animals is the same as identified previously in Saanen goats. Interestingly, analysis of the haplotypes carried by a small set of sex‐reversed animals, known to be associated with polledness, revealed some animals carried the wild‐type chromosome associated with the presence of horns. This suggests a more complex basis for the relationship between polledness and the intersex condition than initially thought while validating the application of the goat SNP50 BeadChip for fine‐mapping traits in goat.     

Combining GWAS and RNA-Seq Approaches for Detection of the Causal Mutation for Hereditary Junctional Epidermolysis Bullosa in Sheep

In this study, we demonstrate the use of a genome-wide association mapping together with RNA-seq in a reduced number of samples, as an efficient approach to detect the causal mutation for a Mendelian disease. Junctional epidermolysis bullosa is a recessive genodermatosis that manifests with neonatal mechanical fragility of the skin, blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions. In Spanish Churra sheep, junctional epidermolysis bullosa (JEB) has been detected in two commercial flocks. The JEB locus was mapped to Ovis aries chromosome 11 by GWAS and subsequently fine-mapped to an 868-kb homozygous segment using the identical-by-descent method. The ITGB4, which is located within this region, was identified as the best positional and functional candidate gene. The RNA-seq variant analysis enabled us to discover a 4-bp deletion within exon 33 of the ITGB4 gene (c.4412_4415del). The c.4412_4415del mutation causes a frameshift resulting in a premature stop codon at position 1472 of the integrin β4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB skin samples and the absence of integrin β4 labeling in immunohistochemical assays. Genotyping of c.4412_4415del showed perfect concordance with the recessive mode of the disease phenotype. Selection against this causal mutation will now be used to solve the problem of JEB in flocks of Churra sheep. Furthermore, the identification of the ITGB4 mutation means that affected sheep can be used as a large mammal animal model for the human form of epidermolysis bullosa with aplasia cutis. Our approach evidences that RNA-seq offers cost-effective alternative to identify variants in the species in which high resolution exome-sequencing is not straightforward.