Proteomic profiling of integral membrane proteins associated to pathogenicity in Vibrio parahaemolyticus strains

Vibrio parahaemolyticus has been recognized as the causal agent of early mortality syndrome and is currently considered an emerging shrimp disease causing losses of millions in the aquaculture industry. Integral membrane proteins are widely recognized as pathogenicity factors involved in essential mechanisms for V. parahaemolyticus infection, which makes them attractive as therapeutic targets. However, their physico‐chemical properties and weak expression has resulted in under‐representation of these proteins in conventional bottom‐up proteomics, making integral membrane proteomics a challenging task. Integral membrane proteins from a bacterial strain isolated from the hepatopancreases of white shrimp with early mortality syndrome and identified by 16S rRNA sequencing as V. parahaemolyticus and an ATCC strain that is pathogenic for humans were obtained by a sequential extraction method and subjected to relative quantification and identification by isobaric Tags for Relative and Absolute Quantitation. A homology database search resulted in identification of more than two hundred proteins, 35 of which are recognized as pathogenic factors showed statistically significant differential accumulation between the strains. These proteins are mainly associated with adherence, secretion systems, cell division, transport, lysogenization, movement and virulence. Identification of pathogenicity‐related proteins in V. parahaemolyticus provides valuable information for developing strategies based on molecular mechanisms that inhibit these proteins, which may be useful therapeutic targets for assisting the shrimp and aquaculture industry.     

Mycotoxin Production by Fusarium Species Isolated from Bananas

The ability of Fusarium species isolated from bananas to produce mycotoxins was studied with 66 isolates of the following species: F. semitectum var. majus (8 isolates), F. camptoceras (3 isolates), a Fusarium sp. (3 isolates), F. moniliforme (16 isolates), F. proliferatum (9 isolates), F. subglutinans (3 isolates), F. solani (3 isolates), F. oxysporum (5 isolates), F. graminearum (7 isolates), F. dimerum (3 isolates), F. acuminatum (3 isolates), and F. equiseti (3 isolates). All isolates were cultured on autoclaved corn grains. Their toxicity to Artemia salina L. larvae was examined. Some of the toxic effects observed arose from the production of known mycotoxins that were determined by thin-layer chromatography, gas chromatography, or high-performance liquid chromatography. All F. camptoceras and Fusarium sp. isolates proved toxic to A. salina larvae; however, no specific toxic metabolites could be identified. This was also the case with eight isolates of F. moniliforme and three of F. proliferatum. The following mycotoxins were encountered in the corn culture extracts: fumonisin B(inf1) (40 to 2,900 (mu)g/g), fumonisin B(inf2) (150 to 320 (mu)g/g), moniliformin (10 to 1,670 (mu)g/g), zearalenone (5 to 470 (mu)g/g), (alpha)-zearalenol (5 to 10 (mu)g/g), deoxynivalenol (8 to 35 (mu)g/g), 3-acetyldeoxynivalenol (5 to 10 (mu)g/g), neosolaniol (50 to 180 (mu)g/g), and T-2 tetraol (5 to 15 (mu)g/g). Based on the results, additional compounds produced by the fungal isolates may play prominent roles in the toxic effects on larvae observed. This is the first reported study on the mycotoxin-producing abilities of Fusarium species that contaminate bananas.     

Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation

Summary Direct hydrolysis of Aspergillus niger mycelium growth on amberlite IRA-900 or sugar cane bagasse on solid state fermentation followed by the analysis of soluble protein by the dye binding method was carried out. Hydrolysis with phosphoric acid 0.25M during 7 min allowed maxima protein extraction available to be measured. Color interference of medium components was not observed, allowing the use of this method for biomass estimation when amberlite IRA-900 or sugar cane bagasse are used as support in solid state fermentation processes.     

Antifungal performance of extracellular chitinases and culture supernatants of <Emphasis Type="Italic">Streptomyces galilaeus</Emphasis> CFFSUR-B12 against <Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis> Morelet

The tropical and mycoparasite strain Streptomyces galilaeus CFFSUR-B12 was evaluated as an antagonist of Mycosphaerella fijiensis Morelet, causal agent of the Black Sigatoka Disease (BSD) of banana. On zymograms of CFFSUR-B12 culture supernatants, we detected four chitinases of approximately 32 kDa (Chi32), 20 kDa (Chi20), and two with masses well over 170 kDa (ChiU) that showed little migration during denaturing electrophoresis at different concentrations of polyacrylamide. The thymol-sulphuric acid assay showed that the ChiU were glycosylated chitinases. Moreover, matrix assisted laser desorption ionization time-of-flight MS analysis revealed that the ChiU are the same protein and identical to a family 18 chitinase from Streptomyces sp. S4 (gi|498328075). Chi32 was similar to an extracellular protein from Streptomyces albus J1074 (gi|478687481) and Chi20 was non-significantly similar to chitinases from five different strains of Streptomyces (P > 0.05). Subsequently, Chi32 and Chi20 were partially purified by anion exchange and hydrophobic interaction chromatography and tested against M. fijiensis. Chitinases failed to inhibit ascospore germination, but inhibited up to 35 and 62 % of germ tube elongation and mycelial growth, respectively. We found that crude culture supernatant and living cells of S. galilaeus CFFSUR-B12 were the most effective in inhibiting M. fijiensis and are potential biocontrol agents of BSD.     

Effects of Extracellular pH on UV-Induced K Efflux from Cultured Rose Cells

Ultraviolet (UV) light causes a specific leakage of K⁺ from cultured rose cells (Rosa damascena). During K⁺ efflux, there is also an increase in extracellular HCO₃⁻ and acidification of the cell interior. We hypothesized that the HCO₃⁻ originated from intracellular hydration of respiratory CO₂ and served as a charge balancing mechanism during K⁺ efflux, the K⁺ and HCO₃⁻ being cotransported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the countertransport of K⁺ and H⁺. To test these hypotheses, we studied the effect of a range of external pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO₂ partial pressure), on the UV-induced K⁺ efflux. Both UV-C (<290 nanometers) and UV-B (290-310 nanometers) induced K⁺ efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K⁺, these data are not consistent with the notion that the efflux of K⁺ is dependent on an influx of H⁺, a process that would be sensitive to external H⁺ concentration. We suggest that the effect of pH on K⁺ efflux may be mediated through the titration of specific K⁺-transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO₂.     

Mycotoxins and mycotoxigenic moulds in nuts and sunflower seeds for human consumption

A survey was carried out to obtain data on the occurrence of mycotoxins and the mycotoxin-producing potential of fungi isolated from nuts (almonds, peanuts, hazelnuts, pistachio nuts) and sunflower seeds in Spain. Thin-layer chromatography was used to separate the toxins. Aflatoxins were detected in one sample of almonds (95 ppb aflatoxin B₁ and 15 ppb aflaxtoxin B₂) and in one sample of peanuts at a level below 10 ppb of aflatoxin B1. 100% of samples showed variable incidence of fungal contamination. The predominant fungi present in samples were Penicillium spp, Aspergillus niger, A. flavus, A. glaucus and Rhizopus spp. The results showed that isolates of different species were able to produce aflatoxins B₁, B₂, G₁, and G₂, sterigmatocystin, ochratoxin A, patulin, citrinin, penicillic acid, zearalenone, and griseofulvin.     

The role of endoscopic ultrasound in the evaluation of rectal cancer

Accurate staging of rectal cancer is essential for selecting patients who can undergo sphincter-preserving surgery. It may also identify patients who could benefit from neoadjuvant therapy. Clinical staging is usually accomplished using a combination of physical examination, CT scanning, MRI and endoscopic ultrasound (EUS). Transrectal EUS is increasingly being used for locoregional staging of rectal cancer. The accuracy of EUS for the T staging of rectal carcinoma ranges from 80-95% compared with CT (65-75%) and MR imaging (75-85%). In comparison to CT, EUS can potentially upstage patients, making them eligible for neoadjuvant treatment. The accuracy to determine metastatic nodal involvement by EUS is approximately 70-75% compared with CT (55-65%) and MR imaging (60-70%). EUS guided FNA may be beneficial in patients who appear to have early T stage disease and suspicious peri-iliac lymphadenopathy to exclude metastatic disease.