Impact of Photocatalysis on Fungal Cells: Depiction of Cellular and Molecular Effects on Saccharomyces cerevisiae

We have investigated the antimicrobial effects of photocatalysis on the yeast model Saccharomyces cerevisiae. To accurately study the antimicrobial mechanisms of the photocatalytic process, we focused our investigations on two questions: the entry of the nanoparticles in treated cells and the fate of the intracellular environment. Transmission electronic microscopy did not reveal any entry of nanoparticles within the cells, even for long exposure times, despite degradation of the cell wall space and deconstruction of cellular compartments. In contrast to proteins located at the periphery of the cells, intracellular proteins did not disappear uniformly. Disappearance or persistence of proteins from the pool of oxidized intracellular isoforms was not correlated to their functions. Altogether, our data suggested that photocatalysis induces the establishment of an intracellular oxidative environment. This hypothesis was sustained by the detection of an increased level of superoxide ions (O₂°⁻) in treated cells and by greater cell cultivability for cells expressing oxidant stress response genes during photocatalytic exposure. The increase in intracellular ROS, which was not connected to the entry of nanoparticles within the cells or to a direct contact with the plasma membrane, could be the result of an imbalance in redox status amplified by chain reactions. Moreover, we expanded our study to other yeast and filamentous fungi and pointed out that, in contrast to the laboratory model S. cerevisiae, some environmental strains are very resistant to photocatalysis. This could be related to the cell wall composition and structure.     

Common polymorphisms of calpain-10 and the risk of polycystic ovary syndrome in Tunisian population: a case–control study

Recent studies have suggested that calpain-10 (CAPN10) gene polymorphisms play a role in the susceptibility to polycystic ovary syndrome (PCOS). The aim of the present study was to evaluate the possible association between three single nucleotide polymorphisms (SNPs) in CAPN10 gene: UCSNP-43 (rs3792267), UCSNP-19 (rs3842570), and UCSNP-63 (rs5030952) and PCOS in Tunisian cases and control women. Study subjects included 127 women with PCOS (mean age 29.8 ± 4.7 year) and 150 healthy women (mean age 33.5 ± 5.6 year). CAPN10 genotyping was carried-out by direct PCR and PCR–RFLP. Linkage disequilibrium pattern in the genomic region explored was determined by HAPLOVIEW 4.2 while reconstruction of haplotypes was done using PHASE 2.1. The phylogenetic distribution of haplotypes in the population was determined by ARLEQUIN 2.000. Six haplotypes were observed. None of SNPs associated with PCOS or its components while the haplotype H4 associated with the phenotype PCOS-obese (P < 0.025). Moreover the pair of haplotypes H1/H4 strongly associated with high blood-pressure (OR = 14.4, P < 0.012). This work confirms the association of CAPN10 gene with metabolic components in PCOS and highlights the role of haplotypes as strong and efficient genetic markers.     

Differential Expression of Alpha 4 Integrins on Effector Memory T Helper Cells during Bordetella Infections. Delayed Responses in Bordetella pertussis

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX) to test the hypothesis that effector memory T-helper cells (emTh) display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i.) with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs) from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7⁺ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4⁺ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease.     

Synthesis and recovery of high bioactive phenolics from table-olive brine process wastewater

The generated wastewater of table-olive brine processing contains a high amount of polyphenols which are endowed with interesting biological activities. The ethyl acetate extract (EAE) of such wastewater shows high hydroxytyrosol (HT) and tyrosol concentrations of 690 and 98 mg g(-1) dry weight extract, respectively. Phenolic compounds analysis was performed by gas chromatography-mass spectrometry (GC-MS). The antioxidant activity was evaluated by the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-Azinobis[3-ethylbenzothiazoline-6-sulfonate] radicals. Total polyphenol content was estimated with the Folin-Ciocalteu assay. Hydroxytyrosol has DPPH and ABTS radicals scavenging activities higher than 2,6-di-tert-butyl-hydroxytoluene (BHT), while triacetylated hydroxytyrosol (triAcHT) was devoid of any antioxidant activity. The assessment of these antioxidant compounds in biological systems was carried out by the determination of their in vitro cytotoxicity against two different human cancer cell lines (HeLa and DG75) and normal peripheral blood lymphocytes (PBL) using the MTT assay. The 50% cytotoxic concentrations were ranged between 27 and 210 microg mL(-1) for Hela and DG75. At the same concentration range, the EAE and the pure HT and triAcHT exhibited an insignificant cytotoxicity against PBL. Incubation of HeLa and DG75 cells with non-cytotoxic concentrations of EAE, HT or triAcHT resulted in a remarkable protection from the oxidative stress induced by Fe(2+). The antimicrobial activity evaluated by the broth dilution NCCL method using Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica) bacteria and (Candida albicans, Aspergillus niger) fungi showed a broad spectrum bactericidal and fungicidal effect of table-olives-EAE, HT and triAcHT. The MICs vary from 125 to 500 microg mL(-1) for bacteria and from 500 to 2000 microg mL(-1) for fungi.     

Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration

Fluid shear stress modulates vascular production of endothelial superoxide anion (O2*-) and nitric oxide (*NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress (tau ave) of 23 dyn/cm2 upregulated Mn-SOD mRNA expression at a higher level than did OSS at tau ave = 0.02 dyn/cm2 +/- 3.0 dyn.cm(-2).s(-1) and at 1 Hz (PSS by 11.3 +/- 0.4-fold vs. OSS by 5.0 +/- 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration.     

Proteolytic breakdown of gliadin by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> isolated from Tunisian fermented dough

The aim of this work was to select strains with proteolytic activity on wheat gliadin, among lactic acid bacteria, previously isolated from Tunisian fermented wheat dough. Hydrolysis of gliadin, as sole nitrogen source, in an agar medium was visualized by a clear zone surrounding colonies. The increase in absorbance due to gliadin breakdown was measured spectrophotometrically using O-phthaldialdehyde (OPA) on Gliadin Glucose Broth medium. Fermented liquid dough inoculated with individual selected Enterococcus faecalis, showed a decrease of the gliadin concentration from 45 g/kg to 18 g/kg determined by sandwich ELISA test (R-7001). Only the enterococci strains show an hydrolysis of gliadin proteins. Strains showing proteolytic activity are gaining more and more importance in cereal based fermented foods and may help to reduce gliadin involved in coeliac disease.     

Molecular Formula and METLIN Personal Metabolite Database Matching Applied to the Identification of Compounds Generated by LC/TOF-MS

In an effort to simplify and streamline compound identification from metabolomics data generated by liquid chromatography time-of-flight mass spectrometry, we have created software for constructing Personalized Metabolite Databases with content from over 15,000 compounds pulled from the public METLIN database (http://metlin.scripps.edu/). Moreover, we have added extra functionalities to the database that (a) permit the addition of user-defined retention times as an orthogonal searchable parameter to complement accurate mass data; and (b) allow interfacing to separate software, a Molecular Formula Generator (MFG), that facilitates reliable interpretation of any database matches from the accurate mass spectral data. To test the utility of this identification strategy, we added retention times to a subset of masses in this database, representing a mixture of 78 synthetic urine standards. The synthetic mixture was analyzed and screened against this METLIN urine database, resulting in 46 accurate mass and retention time matches. Human urine samples were subsequently analyzed under the same analytical conditions and screened against this database. A total of 1387 ions were detected in human urine; 16 of these ions matched both accurate mass and retention time parameters for the 78 urine standards in the database. Another 374 had only an accurate mass match to the database, with 163 of those masses also having the highest MFG score. Furthermore, MFG calculated a formula for a further 849 ions that had no match to the database. Taken together, these results suggest that the METLIN Personal Metabolite database and MFG software offer a robust strategy for confirming the formula of database matches. In the event of no database match, it also suggests possible formulas that may be helpful in interpreting the experimental results.     

Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation system in <Emphasis Type="Italic">Pelargonium graveolens</Emphasis>: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56?±?1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94?±?2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5?±?2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.     

Salt stress reveals differential physiological,biochemical and molecular responses in <Emphasis Type="Italic">T. monococcum</Emphasis> and <Emphasis Type="Italic">T. durum</Emphasis> wheat genotypes

Salt stress responses implicate a complex mechanism and differ from plant species to another. In this study, we analyzed the physiological, biochemical and molecular responses to salt stress of the diploid wheat (T. monococcum) and compared to the tetraploid wheat (T. durum). Our results showed that the diploid wheat cultivar (cv. Turkey) is relatively tolerant to different salt stress conditions than the tetraploid wheat cultivar (cv. Om Rabia3). This tolerance was manifested by significant germination, plant growth and uptake of water generating cell turgor and development. Moreover, total chlorophyll content was higher in the diploid wheat than that in the tetraploid wheat. The Na⁺ content in leaf blade of the cv. Om Rabia3 was significantly higher than that of the cv. Turkey, suggesting that the diploid cultivar accumulates less toxic sodium in the photosynthetic tissues. This mechanism could be explained by the recirculation of the toxic ions Na⁺ into the xylem sap by SOS1 protein, which coordinates with HKT-like proteins to reduce the accumulation of Na⁺ ions in leaf blade. Interestingly, the expression of the three genes SOS1, HKT and NHX was enhanced under salinity especially in leaf blade of the cv. Turkey. Moreover, this wheat cultivar induced the antioxidative enzymes CAT and SOD activity more efficiently than the other cultivar.