The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.     

Surface hydrophobicity, viability and efficacy in biological control of Penicillium oxalicum spores produced in aerial and submerged culture

The surface hydrophobicity, viability and biocontrol ability of Penicillium oxalicum spores, produced either in aerial or submerged culture, were characterized. A phase distribution test showed that spores produced in both methods of culture were highly hydrophobic, but those produced in aerial culture were more hydrophobic. Spores stored fresh at either 4 or 25 degrees C retained a high viability (80%) after 27 weeks of storage, although aerial spores survived better. Freeze-drying severely affected viability, especially of submerged spores. Biocontrol ability against Fusarium oxysporum f. sp. lycopersici was studied in the growth chamber. Aerially- produced spores were more effective than submerged ones. Aerially-produced P. oxalicum spores appeared to have more advantages than those produced by submerged culture, in relation to both viability and efficacy. These results demonstrate that physiological changes occur depending on production conditions which significantly influences quality of the biocontrol agent.     

基于ITS序列分析探讨杜鹃属映山红亚属的组间关系

以叶状苞亚属的叶状苞杜鹃为外类群,以杜鹃属映山红亚属（subg.Tsutsusi)2组12种杜鹃和羊踯躅亚属（subg.Pentanthera）3种4种杜鹃的ITS区（包括5.8S rDNA)的序列了系统学分析。3个亚属的ITS区序长度范围为642－645bp。排序后ITS区的序列长度为653个位点,gap做缺失处理时,变异位点和信息位点分别占6．58％和3．68％。运用PAUP4．0软件分析,获得15个最简树,步长为75,一致性指数（CI）和维持性指数（RI）值分别为0．9333和0．9515,利用15个最简约树获取严格一致树,结果表明：1）映山红亚属为一单系类群,其内部支持率为81％;2）不支持将R.ashiroi独立成假映山红组,也不支持将R.tashiroi并入映山红组,而支持将R.tashiroi并入轮生叶组中的观点;3）支持将R.tsusiophyllum并入映山红组中的观点;4）大字杜鹃的系统位置还需进一步的研究。     

Detection of antigens on nitrocellulose paper immunoblots with monoclonal antibodies

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet (“Western blot”). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.     

Novel structural components of the ventral disc and lateral crest in Giardia intestinalis

Giardia intestinalis is a ubiquitous parasitic protist that is the causative agent of giardiasis, one of the most common protozoan diarrheal diseases in the world. Giardia trophozoites attach to the intestinal epithelium using a specialized and elaborate microtubule structure, the ventral disc. Surrounding the ventral disc is a less characterized putatively contractile structure, the lateral crest, which forms a continuous perimeter seal with the substrate. A better understanding of ventral disc and lateral crest structure, conformational dynamics, and biogenesis is critical for understanding the mechanism of giardial attachment to the host. To determine the components comprising the ventral disc and lateral crest, we used shotgun proteomics to identify proteins in a preparation of isolated ventral discs. Candidate disc-associated proteins, or DAPs, were GFP-tagged using a ligation-independent high-throughput cloning method. Based on disc localization, we identified eighteen novel DAPs, which more than doubles the number of known disc-associated proteins. Ten of the novel DAPs are associated with the lateral crest or outer edge of the disc, and are the first confirmed components of this structure. Using Fluorescence Recovery After Photobleaching (FRAP) with representative novel DAP::GFP strains we found that the newly identified DAPs tested did not recover after photobleaching and are therefore structural components of the ventral disc or lateral crest. Functional analyses of the novel DAPs will be central toward understanding the mechanism of ventral disc-mediated attachment and the mechanism of disc biogenesis during cell division. Since attachment of Giardia to the intestine via the ventral disc is essential for pathogenesis, it is possible that some proteins comprising the disc could be potential drug targets if their loss or disruption interfered with disc biogenesis or function, preventing attachment.     

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16-1 heat-shock gene expression in Caenorhabditis elegans

Recent data suggest that there might be a subtle thermal explanation for the apparent induction by radiofrequency (RF) radiation of transgene expression from a small heat-shock protein (hsp16-1) promoter in the nematode, Caenorhabditis elegans. The RF fields used in the C. elegans study were much weaker (SAR 5-40 mW kg(-1)) than those routinely tested in many other published studies (SAR approximately 2 W kg(-1)). To resolve this disparity, we have exposed the same transgenic hsp16-1::lacZ strain of C. elegans (PC72) to higher intensity RF fields (1.8 GHz; SAR approximately 1.8 W kg(-1)). For both continuous wave (CW) and Talk-pulsed RF exposures (2.5 h at 25 degrees C), there was no indication that RF exposure could induce reporter expression above sham control levels. Thus, at much higher induced RF field strength (close to the maximum permitted exposure from a mobile telephone handset), this particular nematode heat-shock gene is not up-regulated. However, under conditions where background reporter expression was moderately elevated in the sham controls (perhaps as a result of some unknown co-stressor), we found some evidence that reporter expression may be reduced by approximately 15% following exposure to either Talk-pulsed or CW RF fields.     

Lineage-specific evolution and gene flow in Listeria monocytogenes are independent of bacteriophages

Listeria monocytogenes is a foodborne pathogen causing systemic infection with high mortality. To allow efficient tracking of outbreaks a clear definition of the genomic signature of a cluster of related isolates is required, but lineage-specific characteristics call for a more detailed understanding of evolution. In our work, we used core genome MLST (cgMLST) to identify new outbreaks combined to core genome SNP analysis to characterize the population structure and gene flow between lineages. Whilst analysing differences between the four lineages of L. monocytogenes we have detected differences in the recombination rate, and interestingly also divergence in the SNP differences between sub-lineages. In addition, the exchange of core genome variation between the lineages exhibited a distinct pattern, with lineage III being the best donor for horizontal gene transfer. Whilst attempting to link bacteriophage-mediated transduction to observed gene transfer, we found an inverse correlation between phage presence in a lineage and the extent of recombination. Irrespective of the profound differences in recombination rates observed between sub-lineages and lineages, we found that the previously proposed cut-off of 10 allelic differences in cgMLST can be still considered valid for the definition of a foodborne outbreak cluster of L. monocytogenes.     

Arterially perfused neurosphere-derived cells distribute outside the ischemic core in a model of transient focal ischemia and reperfusion in vitro

BACKGROUND: Treatment with neural stem cells represents a potential strategy to improve functional recovery of post-ischemic cerebral injury. The potential benefit of such treatment in acute phases of human ischemic stroke depends on the therapeutic viability of a systemic vascular delivery route. In spite of the large number of reports on the beneficial effects of intracerebral stem cells injection in experimental stroke, very few studies demonstrated the effectiveness of the systemic intravenous delivery approach. METODOLOGY/PRINCIPAL FINDINGS: We utilized a novel in vitro model of transient focal ischemia to analyze the brain distribution of neurosphere-derived cells (NCs) in the early 3 hours that follow transient occlusion of the medial cerebral artery (MCA). NCs obtained from newborn C57/BL6 mice are immature cells with self-renewal properties that could differentiate into neurons, astrocytes and oligodendrocytes. MCA occlusion for 30 minutes in the in vitro isolated guinea pig brain preparation was followed by arterial perfusion with 1x10(6) NCs charged with a green fluorescent dye, either immediately or 60 minutes after reperfusion onset. Changes in extracellular pH and K(+) concentration during and after MCAO were measured through ion-sensitive electrodes. CONCLUSION/SIGNIFICANCE: It is demonstrated that NCs injected through the vascular system do not accumulate in the ischemic core and preferentially distribute in non-ischemic areas, identified by combined electrophysiological and morphological techniques. Direct measurements of extracellular brain ions during and after MCA occlusion suggest that anoxia-induced tissue changes, such as extracellular acidosis, may prevent NCs from entering the ischemic area in our in vitro model of transitory focal ischemia and reperfusion suggesting a role played by the surrounding microenviroment in driving NCs outside the ischemic core. These findings strongly suggest that the potential beneficial effect of NCs in experimental focal brain ischemia is not strictly dependent on their homing into the ischemic region, but rather through a bystander mechanism possibly mediated by the release of neuroprotective factors in the peri-infarct region.     

Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii

IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.