The Aeromonas salmonicida subsp. salmonicida exoproteome: global analysis, moonlighting proteins and putative antigens for vaccination against furunculosis

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Despite the identification of several virulence factors the pathogenesis is still poorly understood. We have used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF5054) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential (GP) and stationary (SP) phases of growth. RESULTS Among the different experimental conditions we obtained semi-quantitative values for a total of 2136 A. salmonicida proteins. Proteins of specific A. salmonicida species were proportionally less detected than proteins common to the Aeromonas genus or those shared with other Aeromonas species, suggesting that in vitro growth did not induce the expression of these genes. Four detected proteins which are unidentified in the genome of reference strains of A. salmonicida were homologous to components of the conjugative T4SS of A. hydrophila pRA1 plasmid. Polypeptides of three proteins which are specific to the 01-B526 strain were also discovered. In supernatants (SNs), the number of detected proteins was higher in SP (326 for wt vs 329 for mutant) than in GP (275 for wt vs 263 for mutant). In pellets, the number of identified proteins (a total of 1536) was approximately the same between GP and SP. Numerous highly conserved cytoplasmic proteins were present in A. salmonicida SNs (mainly EF-Tu, EF-G, EF-P, EF-Ts, TypA, AlaS, ribosomal proteins, HtpG, DnaK, peptidyl-prolyl cis-trans isomerases, GAPDH, Enolase, FbaA, TpiA, Pgk, TktA, AckA, AcnB, Mdh, AhpC, Tpx, SodB and PNPase), and several evidences support the theory that their extracellular localization was not the result of cell lysis. According to the Cluster of Orthologous Groups classification, 29% of excreted proteins in A. salmonicida SNs were currently poorly characterized. CONCLUSIONS In this part of our work we elucidated the whole in vitro exoproteome of hypervirulent A. salmonicida subsp. salmonicida and showed the secretion of several highly conserved cytoplasmic proteins with putative moonlighting functions and roles in virulence. All together, our results offer new information about the pathogenesis of furunculosis and point out potential candidates for vaccine development.

The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Lability of Proteinaceous Material in Estuarine Seston and Subcellular Fractions of Phytoplankton

The rate of proteolysis of amino acids was used to assess the nutritional lability of various materials making up estuarine seston in 3 Maine, USA, estuaries. Physical separations of subcellular fractions of phytoplankton cells led to higher proteolysis rate constants for the cytoplasmic fraction (>1.2 h(-1)) than for the membrane fraction (0.2 to 1 h(-1)). Whole cells, copepod fecal pellets, bottom sediments, and estuarine seston had overlapping ranges of rate constants of 0.17 to 1.3 h(-1), which were indistinguishable from one another. Protein pools in the seston of these estuaries throughout the seasons were dominated by phytoplankton production and its fresh detrital products. Inverse relationships between proteolysis rate constants for estuarine seston and the ratios of pheopigments to chlorophyll indicates that the average lability of seston decreases with the disappearance of cytoplasmic material in suspension. This kinetic approach to the quality of food resources implies the existence of different pools of digestible protein for estuarine heterotrophs with different gut residence times. Preferential enrichment of membrane components in sestonic detritus may result from the differential lability of proteins in cytoplasm versus membrane components of cells.

Feather-pecking response of laying hens to feather and cellulose-based rations fed during rearing.

Recent studies in laying hens have shown that feather peckers eat more feathers than nonpeckers. We hypothesized that food pellets containing feathers would decrease the birds' appetite for feathers and thereby also decrease feather pecking. To separate the effect of feathers from that of insoluble fiber per se, additional control groups were fed pellets containing similar amounts of cellulose. Sixty (experiment 1) and 180 (experiment 2) 1-d-old Lohmann-Selected Leghorn birds were divided into 12 groups of 5 (experiment 1) and 15 (experiment 2) birds, respectively, and kept on slatted floors. During the rearing period, 4 groups each had ad libitum access to either a commercial pelleted diet, a pelleted diet containing 5% (experiment 1) or 10% (experiment 2) of chopped feathers, respectively, or a pelleted diet containing 5% (experiment 1) or 10% (experiment 2) of cellulose, respectively. In the consecutive laying period, all groups received a commercial pelleted diet. In experiment 1, feather pecking was recorded weekly from wk 5 to wk 16. In the laying period, observations were made in wk 18, 20, 22, 23, 24, 25, 26, 27, 28, and 30. In experiment 2, feather pecking was recorded weekly from wk 5 to 11, in wk 16 to wk 18, and in wk 20 and 21. At the end of the rearing period, plumage condition per individual hen was scored. Scores from 1 (denuded) to 4 (intact) were given for each of 6 body parts. The addition of 10% of feathers to the diet reduced the number of severe feather-pecking bouts (P < 0.0129) and improved plumage condition of the back area (P < 0.001) significantly compared with control diets. The relationship between feather pecking/eating and the gastrointestinal consequences thereof, which alter feather pecking-behavior, are unclear. Understanding this relationship might be crucial for understanding the causation of feather pecking in laying hens.

Added value of dual-energy computed tomography versus single-energy computed tomography in assessing ferromagnetic properties of ballistic projectiles: implications for magnetic resonance imaging of gunshot victims.

OBJECTIVE The objective of this study was to assess the discriminative power of dual-energy computed tomography (DECT) versus single-energy CT (SECT) to distinguish between ferromagnetic and non-ferromagnetic ballistic projectiles to improve safety regarding magnetic resonance (MR) imaging studies in patients with retained projectiles. MATERIALS AND METHODS Twenty-seven ballistic projectiles including 25 bullets (diameter, 3-15 mm) and 2 shotgun pellets (2 mm each) were examined in an anthropomorphic chest phantom using 128-section dual-source CT. Data acquisition was performed with tube voltages set at 80, 100, 120, and 140 kV(p). Two readers independently assessed CT numbers of the projectile's core on images reconstructed with an extended CT scale. Dual-energy indices (DEIs) were calculated from both 80-/140-kV(p) and 100-/140-kV(p) pairs; receiver operating characteristics curves were fitted to assess ferromagnetic properties by means of CT numbers and DEI. RESULTS Nine (33%) of the projectiles were ferromagnetic; 18 were nonferromagnetic (67%). Interreader and intrareader correlations of CT number measurements were excellent (intraclass correlation coefficients, >0.906; P<0.001). The DEI calculated from both 80/140 and 100/140 kV(p) were significantly (P<0.05) different between the ferromagnetic and non-ferromagnetic projectiles. The area under the curve (AUC) was 0.75 and 0.8 for the tube voltage pairs of 80/140 and 100/140 kV(p) (P<0.05; 95% confidence interval, 0.57-0.94 and 0.62-0.97, respectively) to differentiate between the ferromagnetic and non-ferromagnetic ballistic projectiles; which increased to 0.83 and 0.85 when shotgun pellets were excluded from the analysis. The AUC for SECT was 0.69 and 0.73 (80 and 100 kV[p], respectively). CONCLUSIONS Measurements of DECT combined with an extended CT scale allow for the discrimination of projectiles with non-ferromagnetic from those with ferromagnetic properties in an anthropomorphic chest phantom with a higher AUC compared with SECT. This study indicates that DECT may have the potential to contribute to MR safety and allow for MR imaging of patients with retained projectiles. However, further studies are necessary before this concept may be used to triage clinical patients before MR.

S100A1 and S100B expression patterns identify differentiation status of human articular chondrocytes.

Many studies in the field of cell-based cartilage repair have focused on identifying markers associated with the differentiation status of human articular chondrocytes (HAC) that could predict their chondrogenic potency. A previous study from our group showed a correlation between the expression of S100 protein in HAC and their chondrogenic potential. The aims of the current study were to clarify which S100 proteins are associated with HAC differentiation status and to provide an S100-based assay for measuring HAC chondrogenic potential. The expression patterns of S100A1 and S100B were investigated in cartilage and in HAC cultured under conditions promoting dedifferentiation (monolayer culture) or redifferentiation (pellet culture or BMP4 treatment in monolayer culture), using characterized antibodies specifically recognizing S100A1 and S100B, by immunohistochemistry, immunocytochemistry, Western blot, and gene expression analysis. S100A1 and S100B were expressed homogeneously in all cartilage zones, and decreased during dedifferentiation. S100A1, but not S100B, was re-expressed in pellets and co-localized with collagen II. Gene expression analysis revealed concomitant modulation of S100A1, S100B, collagen type II, and aggrecan: down-regulation during monolayer culture and up-regulation upon BMP4 treatment. These results strongly support an association of S100A1, and to a lesser extent S100B, with the HAC differentiated phenotype. To facilitate their potential application, we established an S100A1/B-based flow cytometry assay for accurate assessment of HAC differentiation status. We propose S100A1 and S100B expression as a marker to develop potency assays for cartilage regeneration cell therapies, and as a redifferentiation readout in monolayer cultures aiming to investigate stimuli for chondrogenic induction.

Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.

Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes.

Glutathione oxidants such as tertiary butyl hydroperoxide were shown previously to prevent microtubule assembly and cause breakdown of preassembled cytoplasmic microtubules in human polymorphonuclear leukocytes. The objectives of the present study were to determine the temporal relationship between the attachment and ingestion of phagocytic particles and the assembly of microtubules, and simultaneously to quantify the levels of reduced glutathione and products of its oxidation as potential physiological regulators of assembly. Polymorphonuclear leukocytes from human peripheral blood were induced to phagocytize opsonized zymosan at 30 degrees C. Microtubule assembly was assessed in the electron microscope by direct counts of microtubules in thin sections through centrioles. Acid extracts were assayed for reduced glutathione (GSH) and oxidized glutathione (GSSG), by the sensitive enzymatic procedure of Tietze. Washed protein pellets were assayed for free sulfhydryl groups and for mixed protein disulfides with glutathione (protein-SSG) after borohydride splitting of the disulfide bond. Resting cells have few assembled microtubules. Phagocytosis induces a cycle of rapid assembly followed by disassembly. Assembly is initiated by particle contact and is maximal by 3 min of phagocytosis. Disassembly after 5-9 min of phagocytosis is preceded by a slow rise in GSSG and coincides with a rapid rise in protein-SSG. Protein-SSG also increases under conditions in which butyl hydroperoxide inhibits the assembly of microtubules that normally follows binding of concanavalin A to leukocyte cell surface receptors. No evidence for direct involvement of GSH in the induction of assembly was obtained. The formation of protein-SSG, however, emerges as a possible regulatory mechanism for the inhibition of microtubule assembly and induction of their disassembly.

(Table 2) Organic residue at DSDP Hole 76-534A

The kind, sedimentation rate, and diagenesis of organic particles delivered to the North Atlantic seafloor during the Middle Jurassic-Early Cretaceous were responsible for the presence of carbonaceous sediments in Hole 534A. Organic-rich black clays formed from the rapid supply of organic matter; this organic matter was composed of either abundant, well-preserved, and poorly sorted particles of land plants deposited in clays and silty clays within terrigenous turbiditic sequences (tracheal facies) or abundant amorphous debris (xenomorphic facies) generated through the digestive tracts of marine zooplankton and sedimented as fecal pellets. Evidence for the fecal-pellet origin of xenomorphic debris is illustrated. Black clays were also produced in sediments containing less organic matter as a result of the black color of carbonized particles composing all or most of the residues (micrinitic facies). Slowly sedimented hematitic Aptian clays contain very little carbonized, organic debris that survived diagenetic oxidation. In the red calcareous clay sequence of the Late Jurassic, larger amounts of this oxidized debris turned several clay layers black or blackish red. Carbonized debris also dominates the residues recovered in interbedded black and green Albian clays. Carbonization of organic matter in these sediments either turned them black or provided the diagenetic environment for reduced iron. Carbonized debris is also appreciable in burrow-mottled black-green Kimmeridgian clay. The study of Hole 534A organic matter indicates that during the middle Callovian there was a rapid supply of terrigenous organic matter, followed by a late Callovian episode of rapidly supplied xenomorphic debris deposited as fecal pellets. The Late Jurassic-Berriasian was a time of slower sedimentation of organic matter, primarily of a marine dinoflagellate flora in a poorly preserved xenomorphic facies variously affected by diagenetic oxidation. Several intervals of carbonized tracheal tissue in the Oxfordian and Kimmeridgian suggest episodes of oxidized terrigenous matter. The same sequence of Callovian organic events is evident in much of the Early Cretaceous

Faecal pellet production of copepoda collected in water depth up to 30 meters in the eastern Mediterranean Sea in Oktober 2008 during SES_GR2

Mesozooplankton is collected by vertical tows within the Black sea water body mass layer in the NE Aegean, using a WP-2 200 µm net equipped with a large non-filtering cod-end (10 l). Macrozooplankton organisms are removed using a 2000 µm net. A few unsorted animals (approximately 100) are placed inside several glass beaker of 250 ml filled with GF/F or 0.2 µm Nucleopore filtered seawater and with a 100 µm net placed 1 cm above the beaker bottom. Beakers are then placed in an incubator at natural light and maintaining the in situ temperature. After 1 hour pellets are separated from animals and placed in separated flasks and preserved with formalin. Pellets are counted and measured using an inverted microscope. Animals are scanned and counted using an image analysis system. Carbon- Specific faecal pellet production is calculated from a) faecal pellet production, b) individual carbon: Animals are scanned and their body area is measured using an image analysis system. Body volume is then calculated as an ellipsoid using the major and minor axis of an ellipse of same area as the body. Individual carbon is calculated from a carbon- total body volume of organisms (relationship obtained for the Mediterranean Sea by Alcaraz et al. (2003) divided by the total number of individuals scanned and c) faecal pellet carbon: Faecal pellet length and width is measured using an inverted microscope. Faecal pellet volume is calculated from length and width assuming cylindrical shape. Conversion of faecal pellet volume to carbon is done using values obtained in the Mediterranean from: a) faecal pellet density 1,29 g cm**3 (or pg µm**3) from Komar et al. (1981); b) faecal pellet DW/WW=0,23 from Elder and Fowler (1977) and c) faecal pellet C%DW=25,5 Marty et al. (1994).

Northwards and eastwards velocities extracted from a historical run (for the year 2000) of the UVic Earth System Climate Model of Weaver et al. (2001)

This study provides a theoretical assessment of the potential bias due to differential lateral transport on multi-proxy studies based on a range of marine microfossils. Microfossils preserved in marine sediments are at the centre of numerous proxies for paleoenvironmental reconstructions. The precision of proxies is based on the assumption that they accurately represent the overlying watercolumn properties and faunas. Here we assess the possibility of a syn-depositional bias in sediment assemblages caused by horizontal drift in the water column, due to differential settling velocities of sedimenting particles based on their shape, size and density, and due to differences in current velocities. Specifically we calculate the post-mortem lateral transport undergone by planktic foraminifera and a range of other biological proxy carriers (diatoms, radiolaria and fecal pellets transporting coccolithophores) in several regions with high current velocities. We find that lateral transport of different planktic foraminiferal species is minimal due to high settling velocities. No significant shape- or size-dependent sorting occurs before reaching the sediment, making planktic foraminiferal ideal proxy carriers. In contrast, diatoms, radiolaria and fecal pellets can be transported up to 500km in some areas. For example in the Agulhas current, transport can lead to differences of up to 2°C in temperature reconstructions between different proxies in response to settling velocities. Therefore, sediment samples are likely to contain different proportions of local and imported particles, decreasing the precision of proxies based on these groups and the accuracy of the temperature reconstruction.