Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells

Aims The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. Methods and results Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65−/− mouse embryonic fibroblasts reconstituted with non-acetylatable Lys310-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH2-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. Conclusion We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosi

Prozessstufen in der Schweineschlachtung: Einfluss auf den Oberflächenkeimgehalt von Schlachttierkörpern am Beispiel zweier Betriebe

At sequential steps of slaughter, 200 pig carcasses from two abattoirs were examined for total viable counts (TVC), Enterobacteriaceae and coagulase positive Staphylococcus (CPS) by the wet-dry double swab technique at the neck, belly, back, and ham. At abattoir A, mean TVCs (log CFU cm-2) and the proportion of Enterobacteriaceae positive carcasses were reduced after scalding (1.9,12 %), singeing (1.9,66 %) and blast chilling (2.3,17 %), and increased after dehairing (3.4,100 %) and polishing (2.9) (p < 0.05). At abattoir B, mean TVCs (log CFU cm-2) and the proportion of Enterobacteriaceae positive carcasses were reduced after scalding (2.4, 29 %), polishing (3.7) and chilling (2.6; 55 %), and increased after the combined dehairing/singeing (4.7; 97 %) (p < 0.05).The effects of certain process stages were abattoir- and site-specific: (i) at abattoir B, higher results than at abattoir A were obtained after dehairing/singeing and the following process stages, (ii) at abattoir A, the dehairing and polishing processes were important contamination sources, and (iii) for the neck, evisceration and carcass splitting were identified as hygienic weak points. Moreover, scalding, singeing and chilling may be integrated in a risk-based system for pig slaughter, when process parameters are standardized. CPS were found at abattoir A only in few samples after scalding, singeing, and the following process stages. At abattoir B,CPS detection rates increased significantly after dehairing/singeing and remained at a high level. Further investigations and phenotypic and genotypic characterization data are required to evaluate, if a CPS population may have established on the surfaces of the equipment

Aging induces endothelial dysfunction while sparing arterial thrombosis

OBJECTIVE: To assess the effects of aging on arterial thrombus formation by comparing 2-year-old with 11-week-old C57Bl6 mice. METHODS AND RESULTS: Aging is a major risk factor for cardiovascular disease. In humans, assessing the direct effects of aging on vascular homeostasis is difficult because it occurs in the presence of other risk factors. Arterial thrombosis is the critical event in cardiovascular diseases; however, it is not known whether aging per se promotes its occurrence. Mice represent an interesting system to address this issue because they age without spontaneously developing other risk factors. Organ chamber experiments confirmed the advanced level of aging of old mice. As previously shown, old mice exhibited endothelial dysfunction; however, arterial thrombosis induced by photochemical injury was unchanged. Arterial tissue factor expression and activity; expressions of tissue factor pathway inhibitor, thrombomodulin, and plasminogen activator inhibitor 1; prothrombin time; partial thromboplastin time; thrombin-antithrombin complex; and platelet activation were comparable in both groups. CONCLUSIONS: Although these results cannot be directly extrapolated to humans, this study contributes novel important information on the direct effect of aging on arterial thrombosis and underscores the importance of controlling modifiable risk factors in aged individuals

Microbiological contamination of pig and cattle carcasses in different small-scale Swiss abattoirs.

A total of 750 pig carcasses and 535 cattle carcasses from 17 small-scale abattoirs were sampled by excision at four sites (pig: neck, belly, back, ham; cattle: neck, brisket, flank, rump). Samples were examined for total viable counts (TVC) and Enterobacteriaceae. Mean TVCs ranged from 2.4 to 4.2 log10 CFU cm-2 on pig carcasses and from 2.7 to 3.8 log10 CFU cm-2 on cattle carcasses. With regard to EU Regulation (EC) No 2073/2005, TVCs were mainly considered satisfactory (pig: 81.3%; cattle: 71.4%). Amongst sites, the back (pigs) and neck (cattle) tended to yield higher TVCs. Enterobacteriaceae were detected in low counts and on 23.9% of pig carcasses and 21.7% of cattle carcasses. Amongst abattoirs, Enterobacteriaceae prevalence on pig and cattle carcasses ranged from 2.0% to 56.0% and from 0.0% to 55.0%, respectively. Consequently, criteria of the EU Regulation proved to be a suitable tool for the appraisal of microbiological results (TVCs) from pig and cattle carcasses also from small-scale abattoirs. Because the occurrence of Enterobacteriaceae on carcasses was too infrequent to ensure log normality, frequencies should be compared for these organisms

Entomopathogenic pseudomonads can share an insect host with entomopathogenic nematodes and their mutualistic bacteria.

A promising strategy to overcome limitations in biological control of insect pests is the combined application of entomopathogenic pseudomonads (EPPs) and nematodes (EPNs) associated with mutualistic bacteria (NABs). Yet, little is known about interspecies interactions such as competition, coexistence, or even cooperation between these entomopathogens when they infect the same insect host. We investigated the dynamics of bacteria-bacteria interactions between the EPP Pseudomonas protegens CHA0 and the NAB Xenorhabdus bovienii SM5 isolated from the EPN Steinernema feltiae RS5. Bacterial populations were assessed over time in experimental systems of increasing complexity. In vitro, SM5 was outcompeted when CHA0 reached a certain cell density, resulting in the collapse of the SM5 population. In contrast, both bacteria were able to coexist upon haemolymph-injection into Galleria mellonella larvae, as found for three further EPP-NAB combinations. Finally, both bacteria were administered by natural infection routes i.e. orally for CHA0 and nematode-vectored for SM5 resulting in the addition of RS5 to the system. This did not alter bacterial coexistence nor did the presence of the EPP affect nematode reproductive success or progeny virulence. CHA0 benefited from RS5, probably by exploiting access routes formed by the nematodes penetrating the larval gut epithelium. Our results indicate that EPPs are able to share an insect host with EPNs and their mutualistic bacteria without major negative effects on the reproduction of any of the three entomopathogens or the fitness of the nematodes. This suggests that their combination is a promising strategy for biological insect pest control

Genetic ablation of the p66(Shc) adaptor protein reverses cognitive deficits and improves mitochondrial function in an APP transgenic mouse model of Alzheimer's disease

The mammalian ShcA adaptor protein p66(Shc) is a key regulator of mitochondrial reactive oxygen species (ROS) production and has previously been shown to mediate amyloid β (Aβ)-peptide-induced cytotoxicity in vitro. Moreover, p66(Shc) is involved in mammalian longevity and lifespan determination as revealed in the p66(Shc) knockout mice, which are characterized by a 30% prolonged lifespan, lower ROS levels and protection from age-related impairment of physical and cognitive performance. In this study, we hypothesized a role for p66(Shc) in Aβ-induced toxicity in vivo and investigated the effects of genetic p66(Shc) deletion in the PSAPP transgenic mice, an established Alzheimer's disease mouse model of β-amyloidosis. p66(Shc)-ablated PSAPP mice were characterized by an improved survival and a complete rescue of Aβ-induced cognitive deficits at the age of 15 months. Importantly, these beneficial effects on survival and cognitive performance were independent of Aβ levels and amyloid plaque deposition, but were associated with improved brain mitochondrial respiration, a reversal of mitochondrial complex I dysfunction, restored adenosine triphosphate production and reduced ROS levels. The results of this study support a role for p66(Shc) in Aβ-related mitochondrial dysfunction and oxidative damage in vivo, and suggest that p66(Shc) ablation may be a promising novel therapeutic strategy against Aβ-induced toxicity and cognitive impairment.Molecular Psychiatry advance online publication, 19 July 2016; doi:10.1038/mp.2016.112