Potential transfer of aquatic organisms via ballast water with a particular focus on harmful and non-indigenous species: A survey from Adriatic ports.

Ballast water discharges may cause negative impacts to aquatic ecosystems, human health and economic activities by the introduction of potentially harmful species. Fifty untreated ballast water tanks, ten in each port, were sampled in four Adriatic Italian ports and one Slovenian port. Salinity, temperature and fluorescence were measured on board. Faecal indicator bacteria (FIB), phyto- and zooplankton were qualitatively and quantitatively determined to identify the species assemblage arriving in ballast water. FIB exceeded the convention standard limits in 12% of the sampled tanks. Vibrio cholerae was not detected. The number of viable organisms in the size groups (minimum dimension) <50 and ≥10 µm and ≥50 µm resulted above the abundances required from the Ballast Water Management Convention in 55 and 86% of the samples, respectively. This is not surprising as unmanaged ballast waters were sampled. Some potentially toxic and non-indigenous species were observed in both phyto- and zooplankton assemblages.

Bacterial community shift is induced by dynamic environmental parameters in a changing coastal ecosystem (northern Adriatic, northeastern Mediterranean Sea)--a 2-year time-series study.

The potential link between the microbial dynamics and the environmental parameters was investigated in a semi-enclosed and highly dynamic coastal system (Gulf of Trieste, northern Adriatic Sea, NE Mediterranean Sea). Our comprehensive 2-year time-series study showed that despite the shallowness of this area, there was a significant difference between the surface and the bottom bacterial community structure. The bottom bacterial community was more diverse than the surface one and influenced by sediment re-suspension. The surface seawater temperature had a profound effect on bacterial productivity, while the bacterial community structure was more affected by freshwater-borne nutrients and phytoplankton blooms. Phytoplankton blooms caused an increase of Gammaproteobacteria (Alteromonadaceae, SAR86 and Vibrionaceae) and shift in dominance from SAR11 to Rhodobacteraceae taxon at the surface. Our results propose the importance of the water mass movements as drivers of freshwater-borne nutrients and of allochthonous microbial taxa. This study emphasizes the prediction power based on association networks analyses that are fed with long-term measurements of microbial and environmental parameters. These interaction maps offer valuable insights into the response of marine ecosystem to climate- and anthropogenic-driven stressors.

Selective targeting of tumor and stromal cells by a nanocarrier system displaying lipidated cathepsin B inhibitor.

Cathepsin B (CtsB) is a lysosomal cysteine proteinase that is specifically translocated to the extracellular milieu during cancer progression. The development of a lipidated CtsB inhibitor incorporated into the envelope of a liposomal nanocarrier (LNC-NS-629) is described. Ex vivo and in vivo studies confirmed selective targeting and internalization of LNC-NS-629 by tumor and stromal cells, thus validating CtsB targeting as a highly promising approach to cancer diagnosis and treatment.

Stefin B deficiency reduces tumor growth via sensitization of tumor cells to oxidative stress in a breast cancer model.

Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.

Microbial mechanisms coupling carbon and phosphorus cycles in phosphorus-limited northern Adriatic Sea.

The coastal northern Adriatic Sea receives pulsed inputs of riverine nutrients, causing phytoplankton blooms and seasonally sustained dissolved organic carbon (DOC) accumulation-hypothesized to cause episodes of massive mucilage. The underlying mechanisms regulating P and C cycles and their coupling are unclear. Extensive biogeochemical parameters, processes and community composition were measured in a 64-day mesocosms deployed off Piran, Slovenia. We followed the temporal trends of C and P fluxes in P-enriched (P+) and unenriched (P-) mesocosms. An intense diatom bloom developed then crashed; however, substantial primary production was maintained throughout, supported by tightly coupled P regeneration by bacteria and phytoplankton. Results provide novel insights on post-bloom C and P dynamics and mechanisms. 1) Post-bloom DOC accumulation to 186 µM remained elevated despite high bacterial carbon demand. Presumably, a large part of DOC accumulated due to the bacterial ectohydrolytic processing of primary productivity that adventitiously generated slow-to-degrade DOC; 2) bacteria heavily colonized post-bloom diatom aggregates, rendering them microscale hotspots of P regeneration due to locally intense bacterial ectohydrolase activities; 3) Pi turnover was rapid thus suggesting high P flux through the DOP pool (dissolved organic phosphorus) turnover; 4) Alpha- and Gamma-proteobacteria dominated the bacterial communities despite great differences of C and P pools and fluxes in both mesocosms. However, minor taxa showed dramatic changes in community compositions. Major OTUs were presumably generalists adapted to diverse productivity regimes.We suggest that variation in bacterial ectohydrolase activities on aggregates, regulating the rates of POM→DOM transition as well as dissolved polymer hydrolysis, could become a bottleneck in P regeneration. This could be another regulatory step, in addition to APase, in the microbial regulation of P cycle and the coupling between C and P cycles.

Siramesine triggers cell death through destabilisation of mitochondria, but not lysosomes.

A sigma-2 receptor agonist siramesine has been shown to trigger cell death of cancer cells and to exhibit a potent anticancer activity in vivo. However, its mechanism of action is still poorly understood. We show that siramesine can induce rapid cell death in a number of cell lines at concentrations above 20 µM. In HaCaT cells, cell death was accompanied by caspase activation, rapid loss of mitochondrial membrane potential (MMP), cytochrome c release, cardiolipin peroxidation and typical apoptotic morphology, whereas in U-87MG cells most apoptotic hallmarks were not notable, although MMP was rapidly lost. In contrast to the rapid loss of MMP above 20 µM siramesine, a rapid increase in lysosomal pH was observed at all concentrations tested (5-40 µM); however, it was not accompanied by lysosomal membrane permeabilisation (LMP) and the release of lysosomal enzymes into the cytosol. Increased lysosomal pH reduced the lysosomal degradation potential as indicated by the accumulation of immature forms of cysteine cathepsins. The lipophilic antioxidant α-tocopherol, but not the hydrophilic antioxidant N-acetyl-cysteine, considerably reduced cell death and destabilisation of mitochondrial membranes, but did not prevent the increase in lysosomal pH. At concentrations below 15 µM, siramesine triggered cell death after 2 days or later, which seems to be associated with a general metabolic and energy imbalance due to defects in the endocytic pathway, intracellular trafficking and energy production, and not by a specific molecular event. Overall, we show that cell death in siramesine-treated cells is induced by destabilisation of mitochondria and is independent of LMP and the release of cathepsins into the cytosol. Moreover, it is unlikely that siramesine acts exclusively through sigma-2 receptors, but rather through multiple molecular targets inside the cell. Our findings are therefore of significant importance in designing the next generation of siramesine analogues with high anticancer potential.

MAGUKs, scaffolding proteins at cell junctions, are substrates of different proteases during apoptosis.

A major feature of apoptotic cell death is gross structural changes, one of which is the loss of cell-cell contacts. The caspases, executioners of apoptosis, were shown to cleave several proteins involved in the formation of cell junctions. The membrane-associated guanylate kinases (MAGUKs), which are typically associated with cell junctions, have a major role in the organization of protein-protein complexes at plasma membranes and are therefore potentially important caspase targets during apoptosis. We report here that MAGUKs are cleaved and/or degraded by executioner caspases, granzyme B and several cysteine cathepsins in vitro. When apoptosis was induced by UV-irradiation and staurosporine in different epithelial cell lines, caspases were found to efficiently cleave MAGUKs in these cell models, as the cleavages could be prevented by a pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe)fluoromethylketone. Using a selective lysosomal disrupting agent L-leucyl-L-leucine methyl ester, which induces apoptosis through the lysosomal pathway, it was further shown that MAGUKs are also cleaved by the cathepsins in HaCaT and CaCo-2 cells. Immunohistological data showed rapid loss of MAGUKs at the sites of cell-cell contacts, preceding actual cell detachment, suggesting that cleavage of MAGUKs is an important step in fast and efficient cell detachment.

Why some adults with intellectual disability consult their general practitioner more than others.

BACKGROUND: This research identifies factors affecting why some adults with intellectual disability (AWIDs) consult their general practitioner (GP) more than others. Little is known about these factors, despite AWIDs having higher health needs and reduced longevity. Current barriers to accessing health care need to be understood and overcome to achieve improved health outcomes. METHODS: A secondary analysis of data obtained from a stratified randomised sample of AWIDs participating in a cluster randomised trial of hand held health records. The number of GP consultations was obtained retrospectively for the year preceding initial health interviews from GP records. AWIDs and their carers were given separate health interviews using identical/adapted questions where possible. RESULTS: Two hundred and one AWIDs and or their carers from 40 practices participated (response rate 64.6%) with GP consultation data extracted for 187 AWIDs. Overall consulting levels were low, 3.2 per annum for women and 2.2 for men. Increased age, gender (women) and type of carer (paid) were all significantly associated with increased consultations. Carers reporting health problems, medications reported by AWIDs, medications recorded in GP records, and pain reported by AWIDs were also significant factors affecting consultations to GP practices after adjustment for age and type of carer. CONCLUSIONS: Overall consultation rates were lower than expected, and affected by age, gender and type of carer. Targeted interventions are needed to improve attendance and promote health.

Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the Gulf of Trieste.

Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 x 10(9)/L and 2.9 x 10(10)/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the seawater changed at hour intervals in a range from 1.3 x 10(9)/L to 5.1 x 10(9)/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant component of the microbial community in the Gulf of Trieste.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity.

We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Effects of proteinase inhibitors on digestive proteinases and growth of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae).

The physiology of the gut lumen of the red flour beetle, T. castaneum, was studied to determine the conditions for optimal protein hydrolysis. Although the pH of gut lumen extracts from T. castaneum was 6.5, maximum hydrolysis of casein by gut proteinases occurred at pH 4.2. The synthetic substrate N-alpha-benzoyl-DL-arginine-rho-nitroanilide was hydrolyzed by T. castaneum gut proteinases in both acidic and alkaline buffers, whereas hydrolysis of N-succinyl-ala-ala-pro-phe rho-nitroanilide occurred in alkaline buffer. Inhibitors of T. castaneum digestive proteinases were examined to identify potential biopesticides for incorporation in transgenic seed. Cysteine proteinase inhibitors from potato, Job's tears, and sea anemone (equistatin) were effective inhibitors of in vitro casein hydrolysis by T. castaneum proteinases. Other inhibitors of T. castaneum proteinases included leupeptin, L-trans-epoxysuccinylleucylamido [4-guanidino] butane (E-64), tosyl-L-lysine chloromethyl ketone, and antipain. Casein hydrolysis was inhibited weakly by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and soybean trypsin inhibitor (Kunitz). The soybean trypsin inhibitor had no significant effect on growth when it was bioassayed alone, but it was effective when used in combination with potato cysteine proteinase inhibitor. In other bioassays with single inhibitors, larval growth was suppressed by the cysteine proteinase inhibitors from potato, Job's tears, or sea anemone. Levels of inhibition were similar to that observed with E-64, although the moles of proteinaceous inhibitor tested were approximately 1000-fold less. These proteinaceous inhibitors are promising candidates for transgenic seed technology to reduce seed damage by T. castaneum.

Viral abundance and a high proportion of lysogens suggest that viruses are important members of the microbial community in the Gulf of Trieste.

Epifluorescence microscopy and transmission electron microscopy were applied to study virioplankton community in the Gulf of Trieste (northern Adriatic Sea). The total viral abundance was in a range between 2.5 x 10(9)/L and 2.9 x 10(10)/L and was positively correlated with trophic status of the environment. Viruslike particles were significantly correlated with bacterial abundance in all samples studied. Correlations with other physicochemical or biological parameters were not significant. The data suggest that, because of the substantial fraction of tailed viruses present (26%), bacteriophages are an important component of the virioplankton community in the Gulf of Trieste. The abundance of viruslike particles in the seawater changed at hour intervals in a range from 1.3 x 10(9)/L to 5.1 x 10(9)/L. A significant fraction (71%) of the bacterial isolates was inducible in vitro by mitomycin C, and a high occurrence (51%) of lysogenic isolates with more than one phage morphotype present in the lysate was detected. The presence of lysogenic bacteria in the seawater was confirmed in situ with a mitomycin C induction experiment on the natural bacterial population. Results suggest that virioplankton is an abundant component of the microbial community in the Gulf of Trieste.

Amyloid fibril formation by human stefin B: influence of the initial pH-induced intermediate state.

The amyloid fibril field is briefly described, with some stress put on differences between various proteins and possible role for domain swapping. In the main body of the text, first, a short review is given of the folding properties of both human stefins, alpha/beta-type globular proteins of 53% identity with a known three-dimensional fold. Second, in vitro study of amyloid fibril formation by human stefin B (type I cystatin) is described. Solvents of pH 4.8 and pH 3.3 with and without 2,2,2-trifluoroethanol (TFE) were probed, as it has been shown previously that stefin B forms acid intermediates, a native-like and molten globule intermediate, respectively. The kinetics of fibrillation were measured by thioflavin T fluorescence and CD. At pH 3.3, the protein is initially in the molten globule state. The fibrillation is faster than at pH 4.8; however, there is more aggregation observed. On adding TFE at each pH, the fibril formation is further accelerated.