Occurrence of both subspecies of Photobacterium damselae in mullets collected in the river Magra (Italy)

The natural reservoirs and biological characteristics of pathogenic populations of both subspecies of Photobacterium damselae in aquatic habitants remain unclear because of difficulties in obtaining pathogenic strains from the environment. In the present study, we assessed the occurrence of Photobacterium damselae subsp. piscicida and Photobacterium damselae subsp. damselae , considered to be the causative agent of past epizootic outbreaks in mullets collected in the river Magra, Italy. Two hundred and seventy-eight mullets were collected during a period of two years (2008-2009) and analyzed using multiplex PCR. During this period, 57% of fishes were positive for Photobacterium damselae subsp. piscicida and 37% for Photobacterium damselae subsp. damselae, with an higher presence in summer months although none of PCR-positive mullets showed clinical signs of disease. Our results indicate that the two micro-organisms are widespread in the population of mullets studied, and this could be a possible cause for outbreaks in favourable environmental conditions.     

Adipokines,Ghrelin and Obesity‐Associated Insulin Resistance in Nondiabetic Patients with Acute Coronary Syndrome

Altered glucose metabolism negatively modulates outcome in acute coronary syndromes (ACS). Insulin resistance is commonly associated with increasing BMI in the general population and these associations may involve obesity‐related changes in circulating ghrelin and adipokines. We aimed at investigating interactions between BMI, insulin resistance and ACS and their associations with plasma ghrelin and adipokine concentrations. Homeostasis model assessment of insulin resistance (HOMA_IR)‐insulin resistance index, plasma adiponectin, leptin, total (T‐Ghrelin), acylated (Acyl‐Ghrelin), and desacylated ghrelin (Desacyl‐Ghrelin) were measured in 60 nondiabetic ACS patients and 44 subjects without ACS matched for age, sex, and BMI. Compared with non‐ACS, ACS patients had similar HOMA_IR and plasma adipokines, but lower T‐ and Desacyl‐Ghrelin and higher Acyl‐Ghrelin. Obesity (BMI > 30) was associated with higher HOMA_IR, lower adiponectin, and higher leptin (P < 0.05) similarly in ACS and non‐ACS subjects. In ACS (n = 60) HOMA_IR remained associated negatively with adiponectin and positively with leptin independently of BMI and c‐reactive protein (CRP) (P < 0.05). On the other hand, low T‐ and Desacyl‐Ghrelin with high Acyl‐Ghrelin characterized both obese and non‐obese ACS patients and were not associated with HOMA_IR. In conclusion, in ACS patients, obesity and obesity‐related changes in plasma leptin and adiponectin are associated with and likely contribute to negatively modulate insulin resistance. ACS per se does not however enhance the negative impact of obesity on insulin sensitivity. High acylated and low desacylated ghrelin characterize ACS patients independently of obesity, but are not associated with insulin sensitivity.     

Processing of retinal signals in normal and HCN deficient mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1^−/− mice or during a pharmacological blockade of I_h show that, contrary to previous reports, I_kx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I_h and I_kx to the rods'' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.     

New Insights into the Ageing of Linseed Oil Paint Binder: A Qualitative and Quantitative Analytical Study

This paper presents an analytical investigation of paint reconstructions prepared with linseed oil that have undergone typical 19th century treatments in preparation for painting. The oil was mechanically extracted from the same seed lot, which was then processed by various methods: water washing, heat treatments, and the addition of driers, with and without heat. A modern process lead white (Dutch source, Schoonhoven) and a commercially available vine black were used as pigments. The reconstructions were prepared in 1999, and naturally aged from then onwards. We compared thermogravimetric analysis (TG), which yields macromolecular information, with gas chromatography-mass spectrometry (GC-MS) and direct exposure mass spectrometry (DEMS), which both provide molecular information. The study enabled us to quantitatively demonstrate, for the first time, that the parameters used to identify drying oils are deeply influenced by the history of the paint. In particular, here we show that the ratio between the relative amounts of palmitic and stearic acid (P/S), which is used as an index for differentiating between drying oils, is extremely dependent on the pigments present and the age of the paint. Moreover the study revealed that neither the P/S parameter nor the ratios between the relative amounts of the various dicarboxylic acids (azelaic over suberic and azelaic over sebacic) can be used to trace the sorts of pre-treatment undergone by the oil investigated in this study. The final results represent an important milestone for the scientific community working in the field, highlighting that further research is still necessary to solve the identification of drying oils in works of art.     

RalA and RalB Proteins Are Ubiquitinated GTPases, and Ubiquitinated RalA Increases Lipid Raft Exposure at the Plasma Membrane

Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis. One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.     

High yield production process for Shigella outer membrane particles

Gram-negative bacteria naturally shed particles that consist of outer membrane lipids, outer membrane proteins, and soluble periplasmic components. These particles have been proposed for use as vaccines but the yield has been problematic. We developed a high yielding production process of genetically derived outer membrane particles from the human pathogen Shigella sonnei. Yields of approximately 100 milligrams of membrane-associated proteins per liter of fermentation were obtained from cultures of S. sonnei ΔtolR ΔgalU at optical densities of 30-45 in a 5 L fermenter. Proteomic analysis of the purified particles showed the preparation to primarily contain predicted outer membrane and periplasmic proteins. These were highly immunogenic in mice. The production of these outer membrane particles from high density cultivation of bacteria supports the feasibility of scaling up this approach as an affordable manufacturing process. Furthermore, we demonstrate the feasibility of using this process with other genetic manipulations e.g. abolition of O antigen synthesis and modification of the lipopolysaccharide structure in order to modify the immunogenicity or reactogenicity of the particles. This work provides the basis for a large scale manufacturing process of Generalized Modules of Membrane Antigens (GMMA) for production of vaccines from gram-negative bacteria.     

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²⁺ handling relationship in plant cells

The existence of a transport protein that imports cytosolic NAD(+) into peroxisomes has been controversially discussed for decades. Nevertheless, the biosynthesis of NAD(+) in the cytosol necessitates the import of NAD(+) into peroxisomes for numerous reduction/oxidation (redox) reactions. However, a gene encoding such a transport system has not yet been identified in any eukaryotic organism. Here, we describe the peroxisomal NAD(+) carrier in Arabidopsis. Our candidate gene At2g39970 encodes for a member of the mitochondrial carrier family. We confirmed its peroxisomal localization using fluorescence microscopy. For a long time At2g39970 was assumed to represent the peroxisomal ATP transporter. In this study, we could show that the recombinant protein mediated the transport of NAD(+) . Hence, At2g39970 was named PXN for peroxisomal NAD(+) carrier. The loss of PXN in Arabidopsis causes defects in NAD(+) -dependent β-oxidation during seedling establishment. The breakdown of fatty acid released from storage oil was delayed, which led to the retention of oil bodies in pxn mutant seedlings. Based on our results, we propose that PXN delivers NAD(+) for optimal fatty acid degradation during storage oil mobilization.     

Loss of Rb Cooperates with Ras to Drive Oncogenic Growth in Mammalian Cells

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Highlights? Oncogenic Ras drives weak growth through the PI3K canonical growth pathway ? A novel E2F-independent role for the Rb family in cell biogenesis ? Ras and loss of Rb synergize to give robust and sustained cell growth ? PI3K pathway is crucial for sustained growth, acting to maintain nutrient uptake