Uncoupled eNOS annihilates neuregulin-1β-induced cardioprotection: a novel mechanism in pharmacological postconditioning in myocardial infarction

Myocardial infarct size can be limited by pharmacological postconditioning (pPC) with cardioprotective agents. Cardioprotective effects of neuregulin-1β (NRG) via activation of protein kinase B (Akt) and downstream pathways like endothelial nitric oxide synthase (eNOS) have been postulated based on results from cell culture experiments. The purpose of this study was to investigate if eNOS may be involved in pPC with NRG. NRG application in an ex vivo mouse model (C57Bl6) of ischemia–reperfusion injury was analyzed. Unexpectedly, the infarct size increased when NRG was infused starting 5 min prior to reperfusion, even though protective Akt and GSK3β phosphorylation were enhanced. In eNOS deficient mice, however, NRG significantly reduced the infarct size. Co-infusion of NRG and l-arginine (Arg) lead to a reduction in infarct size in wild type animals. Electron paramagnetic resonance measurements revealed that NRG treatment prior to reperfusion leads to an enhanced release of reactive oxygen species compared to controls and this effect is blunted by co-infusion of Arg. This study documents the cardioprotective mechanisms of NRG signaling to be mediated by GSK3β inactivation. This is the first study to show that this protection fails in situations with dysfunctional eNOS. In eNOS deficient mice NRG exerts its protective effect via the GSK3β pathway, suggesting that the eNOS can limit cardioprotection. As dysfunctional eNOS has been described in cardiovascular risk factors like diabetes, hypertension, and hypercholesterolemia these findings can help to explain lack of postconditioning performance in models of cardiovascular co-morbidities.     

Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation

The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ΔΔCq/Normfinder and ΔΔCq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ΔΔCq/Genorm or ΔΔCq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested.     

Mammalian cells acquire epigenetic hallmarks of human cancer during immortalization

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK–ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK–ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.     

Domains of BclA,the major surface glycoprotein of the B. cereus exosporium: glycosylation patterns and role in spore surface properties

The role of the BclA domains of B. cereus ATCC 14579 was investigated in order to understand the phenomena involved in the interfacial processes occurring between spores and inert surfaces. This was done by (i) creating deletions in the collagen-like region (CLR) and the C-terminal domain (CTD) of BclA, (ii) building BclA proteins with various lengths in the CLR and (iii) modifying the hydrophobic upper surface in the CTD. First, it was demonstrated that the CLR was substituted by three residues already reported in the CLR of B. anthracis, viz. rhamnose, 3-O-methyl-rhamnose, and GalNH₂ residues, while the CTD was also substituted by two additional glycosyl residues, viz. 2-O-methyl-rhamnose and 2,4-O-methyl-rhamnose. Regarding the properties of the spores, both CLR and CTD contributed to the adhesion of the spores, which was estimated by measuring the resistance to detachment of spores adhered to stainless steel plates). CLR and CTD also impacted the hydrophobic character and isoelectric point of the spores. It was then shown that the resistance to detachment of the spores was not affected by the physicochemical properties, but by the CLR length and the presence of hydrophobic amino acids on the CTD.     

The histone methyltransferase Dot1 is required for DNA damage repair and proper development in Dictyostelium

Posttranslational histone modifications play an important role in modulating gene expression and chromatin structure. Here we report the identification of histone H3K79 dimethylation in the simple eukaryote Dictyostelium discoideum. We have deleted the D. discoideum Dot1/KMT4 homologue and demonstrate that it is the sole enzyme responsible for histone H3K79me2. Cells lacking Dot1 are reduced in growth and delayed in development, but do not show apparent changes in cell cycle regulation. Furthermore, our results indicate that Dot1 contributes to UV damage resistance and DNA repair in D. discoideum. In summary, the data support the view that the machinery controlling the setting of histone marks is evolutionary highly conserved and provide evidence that D. discoideum is a suitable model system to analyze these modifications and their functions during development and differentiation.     

CYFIP dependent actin remodeling controls specific aspects of Drosophila eye morphogenesis

Cell rearrangements shape organs and organisms using molecular pathways and cellular processes that are still poorly understood. Here we investigate the role of the Actin cytoskeleton in the formation of the Drosophila compound eye, which requires extensive remodeling and coordination between different cell types. We show that CYFIP/Sra-1, a member of the WAVE/SCAR complex and regulator of Actin remodeling, controls specific aspects of eye architecture: rhabdomere extension, rhabdomere terminal web organization, adherens junctions, retina depth and basement membrane integrity. We demonstrate that some phenotypes manifest independently, due to defects in different cell types. Mutations in WAVE/SCAR and in ARP2/3 complex subunits but not in WASP, another major regulator of Actin nucleation, phenocopy CYFIP defects. Thus, the CYFIP-SCAR-ARP2/3 pathway orchestrates specific tissue remodeling processes.     

Which practices co‐deliver food security,climate change mitigation and adaptation,and combat land degradation and desertification?

There is a clear need for transformative change in the land management and food production sectors to address the global land challenges of climate change mitigation, climate change adaptation, combatting land degradation and desertification, and delivering food security (referred to hereafter as “land challenges”). We assess the potential for 40 practices to address these land challenges and find that: Nine options deliver medium to large benefits for all four land challenges. A further two options have no global estimates for adaptation, but have medium to large benefits for all other land challenges. Five options have large mitigation potential (>3 Gt CO₂eq/year) without adverse impacts on the other land challenges. Five options have moderate mitigation potential, with no adverse impacts on the other land challenges. Sixteen practices have large adaptation potential (>25 million people benefit), without adverse side effects on other land challenges. Most practices can be applied without competing for available land. However, seven options could result in competition for land. A large number of practices do not require dedicated land, including several land management options, all value chain options, and all risk management options. Four options could greatly increase competition for land if applied at a large scale, though the impact is scale and context specific, highlighting the need for safeguards to ensure that expansion of land for mitigation does not impact natural systems and food security. A number of practices, such as increased food productivity, dietary change and reduced food loss and waste, can reduce demand for land conversion, thereby potentially freeing‐up land and creating opportunities for enhanced implementation of other practices, making them important components of portfolios of practices to address the combined land challenges.     

Nutrients and water availability constrain the seasonality of vegetation activity in a Mediterranean ecosystem

Anthropogenic nitrogen (N) deposition and resulting differences in ecosystem N and phosphorus (P) ratios are expected to impact photosynthetic capacity, that is, maximum gross primary productivity (GPP_max). However, the interplay between N and P availability with other critical resources on seasonal dynamics of ecosystem productivity remains largely unknown. In a Mediterranean tree–grass ecosystem, we established three landscape‐level (24 ha) nutrient addition treatments: N addition (NT), N and P addition (NPT), and a control site (CT). We analyzed the response of ecosystem to altered nutrient stoichiometry using eddy covariance fluxes measurements, satellite observations, and digital repeat photography. A set of metrics, including phenological transition dates (PTDs; timing of green‐up and dry‐down), slopes during green‐up and dry‐down period, and seasonal amplitude, were extracted from time series of GPP_max and used to represent the seasonality of vegetation activity. The seasonal amplitude of GPP_max was higher for NT and NPT than CT, which was attributed to changes in structure and physiology induced by fertilization. PTDs were mainly driven by rainfall and exhibited no significant differences among treatments during the green‐up period. Yet, both fertilized sites senesced earlier during the dry‐down period (17–19 days), which was more pronounced in the NT due to larger evapotranspiration and water usage. Fertilization also resulted in a faster increase in GPP_max during the green‐up period and a sharper decline in GPP_max during the dry‐down period, with less prominent decline response in NPT. Overall, we demonstrated seasonality of vegetation activity was altered after fertilization and the importance of nutrient–water interaction in such water‐limited ecosystems. With the projected warming‐drying trend, the positive effects of N fertilization induced by N deposition on GPP_max may be counteracted by an earlier and faster dry‐down in particular in areas where the N:P ratio increases, with potential impact on the carbon cycle of water‐limited ecosystems.     

The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion

Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1‐deficient Chlamydia. Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.