Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 anti-apoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins.     

Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution

Hox genes have been implicated in the evolution of many animal body patterns, but the molecular events underlying trait modification have not been elucidated. Pigmentation of the posterior male abdomen is a recently acquired trait in the Drosophila melanogaster lineage. Here, we show that the Abdominal-B (ABD-B) Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. ABD-B regulation of pigmentation evolved through the gain of ABD-B binding sites in a specific cis-regulatory element of the yellow gene of a common ancestor of sexually dimorphic species. Within the melanogaster species group, male-specific pigmentation has subsequently been lost by at least three different mechanisms, including the mutational inactivation of a key ABD-B binding site in one lineage. These results demonstrate how Hox regulation of traits and target genes is gained and lost at the species level and have general implications for the evolution of body form at higher taxonomic levels.     

Evidence for Geographic Isolation and Signs of Endemism within a Protistan Morphospecies

The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of “Spumella-like” flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.     

Platelets orchestrate remote tissue damage after mesenteric ischemia-reperfusion

Ischemia-reperfusion (I/R) injury is a leading cause of morbidity and mortality. A functional role for platelets in tissue damage after mesenteric I/R is largely unknown. The hypothesis that mesenteric I/R local and remote injury are platelet dependent was tested. Using a murine mesenteric I/R model, we demonstrate that platelets orchestrate remote lung tissue damage that follows mesenteric I/R injury and also contribute, albeit to a lesser degree, to local villi damage. While lung damage is delayed compared with villi damage, it increased over time and was characterized by accumulation of platelets in the pulmonary vasculature early, followed by alveolar capillaries and extravasation into the pulmonary space. Both villi and lung tissues displayed complement deposition. We demonstrate that villi and lung damage are reduced in mice made platelet deficient before I/R injury and that platelet transfusion into previously platelet-depleted mice before I/R increased both villi and lung tissue damage. Increased C3 deposition accompanied platelet sequestration in the lung, which was mostly absent in platelet-depleted mice. In contrast, C3 deposition was only minimally reduced on villi of platelet-depleted mice. Our findings position platelets alongside complement as a significant early upstream component that orchestrates remote lung tissue damage after mesenteric I/R and strongly suggest that reperfusion injury mitigating modalities should consider the contribution of platelets.     

Platelet-associated CD40/CD154 mediates remote tissue damage after mesenteric ischemia/reperfusion injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage.     

miR-Sens--a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

MicroRNA-mRNA interactions are commonly validated and deconstructed in cell lines transfected with luciferase reporters. However, due to cell type-specific variations in microRNA or RNA-binding protein abundance, such assays may not reliably reflect microRNA activity in other cell types that are less easily transfected. In order to measure miRNA activity in primary cells, we constructed miR-Sens, a MSCV-based retroviral vector that encodes both a Renilla luciferase reporter gene controlled by microRNA binding sites in its 3' UTR and a Firefly luciferase normalization gene. miR-Sens sensors can be efficiently transduced in primary cells such as human fibroblasts and mammary epithelial cells, and allow the detection of overexpressed and, more importantly, endogenous microRNAs. Notably, we find that the relative luciferase activity is correlated to the miRNA expression, allowing quantitative measurement of microRNA activity. We have subsequently validated the miR-Sens 3' UTR vectors with known human miRNA-372, miRNA-373, and miRNA-31 targets (LATS2 and TXNIP). Overall, we observe that miR-Sens-based assays are highly reproducible, allowing detection of the independent contribution of multiple microRNAs to 3' UTR-mediated translational control of LATS2. In conclusion, miR-Sens is a new tool for the efficient study of microRNA activity in primary cells or panels of cell lines. This vector will not only be useful for studies on microRNA biology, but also more broadly on other factors influencing the translation of mRNAs.     

Two different secondary metabolism gene clusters occupied the same ancestral locus in fungal dermatophytes of the arthrodermataceae

Background

Dermatophyte fungi of the family Arthrodermataceae (Eurotiomycetes) colonize keratinized tissue, such as skin, frequently causing superficial mycoses in humans and other mammals, reptiles, and birds. Competition with native microflora likely underlies the propensity of these dermatophytes to produce a diversity of antibiotics and compounds for scavenging iron, which is extremely scarce, as well as the presence of an unusually large number of putative secondary metabolism gene clusters, most of which contain non-ribosomal peptide synthetases (NRPS), in their genomes. To better understand the historical origins and diversification of NRPS-containing gene clusters we examined the evolution of a variable locus (VL) that exists in one of three alternative conformations among the genomes of seven dermatophyte species.

Results

The first conformation of the VL (termed VLA) contains only 539 base pairs of sequence and lacks protein-coding genes, whereas the other two conformations (termed VLB and VLC) span 36 Kb and 27 Kb and contain 12 and 10 genes, respectively. Interestingly, both VLB and VLC appear to contain distinct secondary metabolism gene clusters; VLB contains a NRPS gene as well as four porphyrin metabolism genes never found to be physically linked in the genomes of 128 other fungal species, whereas VLC also contains a NRPS gene as well as several others typically found associated with secondary metabolism gene clusters. Phylogenetic evidence suggests that the VL locus was present in the ancestor of all seven species achieving its present distribution through subsequent differential losses or retentions of specific conformations.

Conclusions

We propose that the existence of variable loci, similar to the one we studied, in fungal genomes could potentially explain the dramatic differences in secondary metabolic diversity between closely related species of filamentous fungi, and contribute to host adaptation and the generation of metabolic diversity.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.