Generierung und funktionelle in vivo Charakterisierung einer Lipidkinasedefekten Phosphatidylinositol 3-Kinase CaVps34p der humanpathogenen Hefe Candida albicans

Durch Modifikation einer vermuteten PI-Kopfgruppeninteraktionsregion der multifunktionellen PI 3-Kinase CaVps34p aus Candida albicans konnte eine CaVps34T-Proteinmutante generiert werden, bei der die Lipidkinaseaktivität ausgeschalten wurde, ohne die Proteinkinaseaktivität zu beeinflussen. Nach Herstellung einer Lipidkinase-defekten VPS34 C. albicans-Mutante mit mutiertem VPS34T-Allel wurde gezeigt, dass die Lipidkinaseaktivität von CaVps34p essentiell für den Vesikeltransport zur Vakuole und die Vakuolenansäuerung ist, aber auch die Hyphenbildung und Adhäsion von C. albicans an Wirtszellen beeinflusst sowie letztlich entscheidend für die Virulenz des Pilzes ist. Unabhängig von der Lipidkinaseaktivität beeinflusst CaVps34p Entgiftungsmechanismen der Hefezelle, wobei vermutet wird, dass dabei die Interaktion von CaVps34p mit Vma7p, der Untereinheit der vakuolären ATPase, sowie mit potentiellen Membrantranslokatoren eine Rolle spielt

Moderation of alcohol consumption as a recommendation in European hypertension management guidelines: a survey on awareness, screening and implementation among European physicians

Objectives: Moderation of alcohol consumption is included as a class I, level of evidence A recommendation in the current European guidelines for the management of hypertension. We investigated its awareness and self-reported implementation among European physicians across different specialties and workplaces. Design and setting: A cross-sectional survey study conducted in two annual German meetings (German Society of Cardiology and the German Society of Internal Medicine) and two annual European meetings (European Society of Hypertension and European Society Cardiology) in 2015. Participants: 1064 physicians attending the European meetings were interviewed including 52.1% cardiologists, 29.2% internists and 8.8% general practitioners. Main outcome measures: Physician screening of alcohol consumption, awareness and self-implementation of the recommendation of the current European guidelines about moderation of alcohol consumption for the management of hypertension. Results: Overall, 81.9% of physicians reported to generally quantify alcohol consumption in patients with hypertension. However, only 28.6% and 14.5% of participants reported screening alcohol consumption in their patients with newly detected or treatment-resistant hypertension. Physicians recommended a maximum alcohol intake of 13.1 +/- 11.7 g/day for women (95% CI 12.3 to 13.8) and 19.9 +/- 15.6 g/day for men (95% CI 18.8 to 20.9). In case of moderate to high alcohol consumption, 10.3% would manage only hypertension without addressing alcohol consumption, while 3.7% of the physicians would do so in case of alcohol dependence (p<0.001). Conclusions: The average amount of alcohol intake per day recommended by European physicians in this survey was in agreement with the guidelines. The low number of physicians that screen for alcohol consumption in patients with newly detected and with treatment-resistant hypertension indicates an important deficit in the management of hypertension

TLR ligands, but not modulators of histone modifiers, can induce the complex immune response pattern of endotoxin tolerance in mammary epithelial cells

Excessive stimulation of the TLR4 axis through LPS reduces the expression of some cytokine genes in immune cells, while stimulating the expression of immune defense genes during a subsequent bacterial infection. This endotoxin tolerance (ET) is mediated via epigenetic mechanisms. Priming the udder of cows with LPS was shown to induce ET in mammary epithelial cells (MEC), thereby protecting the udder against reinfection for some time. Seeking alternatives to LPS priming we tried to elicit ET by priming MEC with either lipopeptide (Pam2CSK4) via the TLR2/6 axis or inhibitors of histone-modifying enzymes. Pre-incubation of MEC with Pam2CSK4 enhanced baseline and induced expression of bactericidal (beta-defensin;SLPI) and membrane protecting factors (SAA3, TGM3), while reducing the expression of cytokine-and chemokine-encoding genes (TNF, IL1 beta) after a subsequent pathogen challenge, the latter, however, not as efficiently as after LPS priming. Pre-treating MEC with various inhibitors of histone H3 modifiers (for demethylation, acetylation or deacetylation) all failed to induce any of the protective factors and only resulted in some dampening of cytokine gene expression after the re-challenge. Hence, triggering immune functions via the TLR axis, but not through those histone modifiers, induced the beneficial phenomenon of ET in MEC

A Practical Second-Order Fault Attack against a Real-World Pairing Implementation

Several fault attacks against pairing-based cryptography have been described theoretically in recent years. Interestingly, none of these have been practically evaluated. We accomplished this task and prove that fault attacks against pairing-based cryptography are indeed possible and are even practical — thus posing a serious threat. Moreover, we successfully conducted a second-order fault attack against an open source implementation of the eta pairing on an AVR XMEGA A1. We injected the first fault into the computation of the Miller Algorithm and applied the second fault to skip the final exponentiation completely. We introduce a low-cost setup that allowed us to generate multiple independent faults in one computation. The setup implements these faults by clock glitches which induce instruction skips. With this setup we conducted the first practical fault attack against a complete pairing computation

Early transcriptional events in the udder and teat after intra-mammary Escherichia coli and Staphylococcus aureus challenge

Intra-mammary bacterial infections can result in harmful clinical mastitis or subclinical mastitis with persistent infections. Research during the last decades closely examined the pathophysiology of inflamed udders. Initial events after pathogen perception but before the onset of mastitis have not been examined invivo. The objective of this study was to develop a mastitis model in cows by monitoring initial transcriptional pathogen-specific host response before clinical signs occur. We applied a short-term infection model to analyse transcripts encoding chemokines, cytokines and antimicrobial molecules in the teat cistern (TC) and lobulo-alveolar parenchyma (LP) up to 3h after challenge with E. and Staphylococcus aureus. Both pathogens elicited an immune reaction by 1h after challenge. Escherichia coli induced all analysed factors (CCL20, CXCL8, TNF, IL6, IL12B, IL10, LAP, S100A9);however, S. aureus failed to induce IL12B, IL10, LAP and S100A9 expression. The E. coli-induced up-regulation was 25-105 times greater than that after S. aureus challenge. Almost all the responses were restricted to the TC. The short-term mastitis model demonstrates that a divergent pathogen-specific response is generated during the first h. It confirms that the first transcripts are generated in the TC prior to a response in the LP

Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia coli

We examined the repertoire and extent of inflammation dependent gene regulation in a bovine mammary epithelial cell (MEC) model, to better understand the contribution of the MEC in the immune defence of the udder. We challenged primary cultures of MEC from cows with heat inactivated Escherichia coli pathogens and used Affymetrix DNA-microarrays to profile challenge related alterations in their transcriptome. Compared to acute mastitis, the most prominently activated genes comprise those encoding chemokines, interleukins, beta-defensins, serum amyloid A and haptoglobin. Hence, the MEC exert sentinel as well as effector functions of innate immune defence. E. coli stimulated a larger fraction of genes (30%) in the MEC belonging to the functional category Inflammatory Response than we recorded with the same microarrays during acute mastitis in the udder (17%). This observation underscores the exquisite immune capacity of MEC. To more closely examine the adequacy of immunological regulation in MEC, we compared the inflammation dependent regulation of factors contributing to the complement system between the udder versus the MEC. In the MEC we observed only up regulation of several complement factor-encoding genes. Mastitis, in contrast, in the udder strongly down regulates such genes encoding factors contributing to both, the classical pathway of complement activation and the Membrane Attack Complex, while the expression of factors contributing to the alternative pathway may be enhanced. This functionally polarized regulation of the complex complement pathway is not reflected in the MEC models

Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response

Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant Staphylococcus aureus infections in Europe and North America. This strain is characterised by high promiscuity in terms of host species and also lacks several traditional S. aureus virulence factors. This does not however explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) inhibitng the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain Domain of Unknown Function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed the potential for a slightly increased survival rate in a murine infectious model compared to the wild-type strain at one dose, but the data did not reach level of significance. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers

Remote sensing of drained thermokarst lake basin successions

Thermokarst lakes are important factors for permafrost landscape dynamics and carbon cycling. Thermokarst lake cover is particularly high in Arctic lowlands with ice-rich permafrost. In many of these vast lowland regions, drained thermokarst lake basins of different age have been identified that overlap each other in space, suggesting intense dynamics of repeated lake formation and loss with complex carbon cycle histories during the Holocene [Grosse et al.,2013]. Observing the permafrost and ecosystem succession patterns following thermokarst lake drainage will help to better determining the landscape and regional scale impacts of lake loss on northern hydrology, permafrost aggradation, vegetation succession, carbon cycling, as well as spectral land surface property changes. Previous remote sensing approaches to study drained thermokarst lake basins used a combination of Landsat, high resolution satellite and aerial, and field data to quantify carbon stocks accumulated in post-drainage peat in drained thermokarst lake basins (DTLBs) for the northern Seward Peninsula in Northwest Alaska [Jones et al., 2012]. We here expand on this method by using different remote sensing products in combination with dating of lake drainage events. These events are identified based on the historical remote sensing record and accelerated mass spectrometry radiocarbon dating. The joint us of remote sensing and geochronological field data allows to assess the specific succession patterns of various DTLB types and their impacts on land surface properties in different Arctic permafrost regions (North Alaska, Northwest Alaska, North Siberia). The datasets used in this analysis include a range of remote sensing and topographic data, such as aerial photography, historic topographic maps, high resolution satellite images (Corona, Spot, Ikonos, Quickbird, Worldview, GeoEye), and imagery from the full Landsat archive as well as from the Moderate Resolution Imaging Spectrometer (MODIS) sensors. We report temporal trends of spectral properties based on Landsat multispectral indices for individual DTLBs of different ages, but also employ landscape-scale chronosequences allowing the analysis succession trajectories of DTLBs that drained well before the start of the remote sensing record. Here we are particularly focusing on the long-term impacts of lake drainage on changes in normalized difference vegetation index (NDVI), normalized difference moisture index (NDMI), normalized difference water index (NDWI), Tasseled Cap index (brightness, greenness, wetness), land surface temperatures, and albedo. We further conducted field studies including reconnaissance flights targeting historically drained lakes and cored DTLBs to sample for radiocarbon-dating of terrestrial peat layers indicative of the drainage event. Results of this ongoing study suggest a strong impact exerted by thermokarst lake drainage on land surface reflectance characteristics in thermokarst lowland regions. The information may be useful for parameterizing surface properties in land surface models of thermokarst-affected regions, particularly where increased lake drainage is projected to take place

<em>Escherichia coli</em>- and <em>Staphylococcus aureus </em>- induced mastitis differentially modulate transcriptional responses in neighbouring uninfected bovine mammary gland quarters

BACKGROUND: The most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared. RESULTS: The bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows. CONCLUSIONS: Uninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland