Proteomic analysis of phytopathogenic fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis> as a potential tool for identifying pathogenicity factors,therapeutic targets and for basic research

Botrytis cinerea is a phytopathogenic fungus causing disease in a substantial number of economically important crops. In an attempt to identify putative fungal virulence factors, the two-dimensional gel electrophoresis (2-DE) protein profile from two B. cinerea strains differing in virulence and toxin production were compared. Protein extracts from fungal mycelium obtained by tissue homogenization were analyzed. The mycelial 2-DE protein profile revealed the existence of qualitative and quantitative differences between the analyzed strains. The lack of genomic data from B. cinerea required the use of peptide fragmentation data from MALDI-TOF/TOF and ESI ion trap for protein identification, resulting in the identification of 27 protein spots. A significant number of spots were identified as malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The different expression patterns revealed by some of the identified proteins could be ascribed to differences in virulence between strains. Our results indicate that proteomic analysis are becoming an important tool to be used as a starting point for identifying new pathogenicity factors, therapeutic targets and for basic research on this plant pathogen in the postgenomic era.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

Lipids and DNA oxidation in Staphylococcus aureus as a consequence of oxidative stress generated by ciprofloxacin

Ciprofloxacin induced an increment of reactive oxygen species in sensitive strains of Staphylococcus aureus leading to oxidative stress detected by chemiluminescence while resistant strains did not suffer such stress. Oxidation of lipids was performed by employing thiobarbituric acid reaction to detect the formation of the amplified intermediate between reactive species oxygen and cytoplasmic macromolecules, namely malondialdehyde (MDA). The sensitive strain presented higher peroxidation of lipids than the resistant strain. The oxidative consequence for DNA was investigated by means of bacteria incubation with ciprofloxacin and posterior extraction of DNA, which was studied by high performance liquid chromatography (HPLC). Sensitive S. aureus ATCC 29213 showed an increase of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) respect controls without antibiotic; there was evident increase of the ratio between 8-oxodG and deoxyguanosine (dG) as a consequence of oxidation of dG to 8-oxodG considered the major DNA marker of oxidative stress. The resistant strain showed low oxidation of DNA and the analysis of 8-oxodG/dG ratio indicated lesser formation of 8-oxodG than S. aureus ATCC 29213.     

Biofilms of As(III)-oxidising bacteria: formation and activity studies for bioremediation process development

The formation and activity of an As(III)-oxidising biofilm in a bioreactor, using pozzolana as bacterial growth support, was studied for the purpose of optimising fixed-bed bioreactors for bioremediation. After 60 days of continuous functioning with an As(III)-contaminated effluent, the active biofilm was found to be located mainly near the inflow rather than homogeneously distributed. Biofilm development by the CAsO1 bacterial consortium and by Thiomonas arsenivorans was then studied both on polystyrene microplates and on pozzolana. Extra-cellular polymeric substances (EPS) and yeast extract were found to enhance bacteria attachment, and yeast extract also appears to increase the kinetics of biofilm formation. Analysis of proteins, sugars, lipids and uronic acids indicate that sugars were the main EPS components. The specific As(III)-oxidase activity of T. arsenivorans was higher (by ninefold) for planktonic cells than for sessile ones and was induced by As(III). All the results suggest that the biofilm structure is a physical barrier decreasing As(III) access to sessile cells and thus to As(III)-oxidase activity induction. The efficiency of fixed-bed reactors for the bioremediation of arsenic-contaminated waters can be thus optimised by controlling different factors such as temperature and EPS addition and/or synthesis to increase biofilm density and activity.     

Modified SureSelect<Superscript>QXT</Superscript> Target Enrichment Protocol for Illumina Multiplexed Sequencing of FFPE Samples

Background

Personalised medicine is nowadays a major objective in oncology. Molecular characterization of tumours through NGS offers the possibility to find possible therapeutic targets in a time- and cost-effective way. However, the low quality and complexity of FFPE DNA samples bring a series of disadvantages for massive parallel sequencing techniques compared to high-quality DNA samples (from blood cells, cell cultures, etc.).

Results

We performed several experiments to understand the behaviour of FFPE DNA samples during the construction of SureSelect^QXT libraries. First, we designed a quality checkpoint for FFPE DNA samples based on the quantification of their amplification capability (qcPCR). We observed that FFPE DNA samples can be classified according to DIN value and qcPCR concentration into unusable, or low-quality (LQ) and good-quality (GQ) DNA. For GQ samples, we increased the amount of input DNA to 150 ng and the digestion time to 30 min, whereas for LQ samples, we used 50 ng of DNA as input but we decreased the digestion time to 1 min. In all cases, we increased the cycles of the pre-hyb PCR to 10 but decreased the cycles of the post-hyb PCR to 8. In addition, we confirmed that using half of the volume of reagents can be beneficial. Finally, in order to obtain better results, we designed a decision flow-chart to achieve a seeding concentration of 12–14 pM for MiSeq Reagent Kit v2.

Conclusions

Our experiments allowed us to unveil the behaviour of low-quality FFPE DNA samples during the construction of SureSelect^QXT libraries. Sequencing results showed that, using our modified SureSelect^QXT protocol, the final percentage of usable reads for low-quality samples was increased more than three times allowing to reach median depth/million reads values of 76.35. This value is equivalent to ~?0.9 and ~?0.7 of the values obtained for good-quality FFPE and high-quality DNA respectively.

     

Effects of Estrogen and Phytoestrogen Treatment on an In Vitro Model of Recurrent Stroke on HT22 Neuronal Cell Line

An increase of stroke incidence occurs in women with the decline of estrogen levels following menopause. This ischemic damage may recur, especially soon after the first insult has occurred. We evaluated the effects of estrogen and phytoestrogen treatment on an in vitro recurrent stroke model using the HT22 neuronal cell line. HT22 cells were treated with 17β-estradiol or genistein 1 h after the beginning of the first of two oxygen and glucose deprivation/reoxygenation (OGD/R) cycles. During the second OGD, there was a deterioration of some components of the electron transport chain, such as cytochrome c oxidase subunit 1 with a subsequent increase of reactive oxygen species (ROS) production. Accordingly, there was also an increase of apoptotic phenomena demonstrated by poly(ADP-ribose) polymerase 1 cleavage, Caspase-3 activity, and Annexin V levels. The recurrent ischemic injury also raised the hypoxia-inducible factor 1α and glucose transporter 1 levels, as well as the ratio between the lipidated and cytosolic forms of microtubule-associated protein 1A/1B-light chain 3 (LC3-II/LC3-I). We found a positive effect of estradiol and genistein treatment by partially preserving the impaired cell viability after the recurrent ischemic injury; however, this positive effect does not seem to be mediated neither by blocking apoptosis processes nor by decreasing ROS production. This work contribute to the better understanding of the molecular mechanisms triggered by recurrent ischemic damage in neuronal cells and, therefore, could help with the development of an effective treatment to minimize the consequences of this pathology.     

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.