T, was characterized using a polyphasic approach comprising phylogenetic, morphological and genotypic analyses. The endophytic strain was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. Analysis of the 16S rRNA gene demonstrated that the novel strain is most closely related to members of the genus Spirosoma . The genomic DNA G+C content was 47.2 mol%. The predominant quinone was and the major cellular fatty acids were summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0. On the basis of its phenotypic and genotypic properties, strain EX36T should be classified as a novel species of the genus Spirosoma, for which the name Spirosomaendophyticum sp. nov. is proposed. The type strain is EX36T ( = DSM 26130T = LMG 27272T).A Gram-reaction-negative, yellow-pigmented strain, designated EX36 Spirosoma was first proposed by Flexibacteraceae in the phylum Bacteroidetes. At the time of writing the genus Spirosoma includes five species, the type species Spirosoma linguale  were sampled  containing 15 % glycerol and stored at −80 °C. Endophytic colonization was confirmed by inoculating two maize and two potato cultivars, growing the plants under in vitro conditions and reisolating the strain from root and stem tissues.For the isolation of strain EX36seqmanpro (DNAstar). The consensus sequence was subjected to nucleotide blast analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to search the database of the National Center for Biotechnology Information (NCBI) for the closest relatives of the bacterial strains with validly published names. Sequence comparisons indicated that the isolate belonged to the family Flexibacteraceae.For the extraction of bacterial DNA the Gen Elute Bacterial Genomic DNA kit (Sigma–Aldrich) was used. The 16S rRNA gene was amplified by PCR using the primers 8f (5′-AGAGTTTGATCCTGGCTCAG-3′)  and seqmT and of all species of the genus Spirosoma with validly published names and of selected species of the family Cytophagaceae, which were downloaded from the NCBI GenBank sequence database, were imported into the arb program package , followed by S. luteum SPM-10T (93.9 %), S. spitsbergense SPM-9T (93.9 %), S. rigui KCTC 12531T (93.8 %) and S. panaciterrae Gsoil 1519T (92.5 %).Nearly complete 16S rRNA gene sequences of strain EX36v. 7.4.2  by execuv. 7.4.2 : raxmlHPv. 7.4.2  and the v. 7.4.2 , which wlgorithm , which wT was tested at various temperatures  on 10 % TSA plates for up to 1 week. The pH range for growth  was determined by measuring OD600 changes in cultures incubated at 28 °C with shaking at 190 r.p.m. compared with an uninoculated control. Salt tolerance was determined by amending 10 % TSB with NaCl to final concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0 and 10.0 % NaCl (w/v). The Gram reaction of strain EX36T was determined by using the non-staining method described by Growth of strain EX36T were rod-shaped, Gram-reaction-negative and 1.2×2−17.5 µm in size . Most cells were arranged in pairs, but filaments up to 55 µm were observed. EX36T showed yellowish, opaque, semi-translucent colonies with a smooth and shiny surface and a circular and convex shape. The diameter of colonies grown on 10 % TSA at 28 °C for 1 week varied between 1.5 and 3.0 mm. The strain was positive for catalase and oxidase activity; detailed results of biochemical and physiological analyses are listed in Spirosoma, cells of EX36T showed a length up to 17.5 µm, did not grow at 5 and 42 °C, did not tolerate NaCl concentrations higher than 0.6 % (w/v), had the lowest genomic G+C content and showed differences in antibiotic susceptibility. Low tolerance of Cd and Zn was observed (slow growth at 4 mM Zn and 1 mM Cd). The analysis of yellow pigments showed three absorption maxima at 428, 453 and 483 nm. EX36T was negative for flexirubin-type pigments.Cells of strain EX36T were summed feature 3 , C16 : 1ω5c (23.8 %), iso-C17 : 0 3-OH (6.2 %) and iso-C15 : 0 (5.4 %). A detailed overview of the cellular fatty acid profiles of all species of the genus Spirosoma can be found in T and other species of the genus Spirosoma were found in the amounts of iso-C15 : 0, C16 : 1ω5c and summed feature 3. In contrast to S. linguale DSM 74T, the fatty acids C15 : 0 and anteiso-C15 : 0 were not detected.Analyses of cellular fatty acid composition, respiratory quinones, polar lipids and chromosomal G+C content were performed by the Identification Service of the DSMZ. The fatty acid profile was determined according to the protocol of the Microbial Identification System (MIDI). The major fatty acids of strain EX36Spirosoma, was MK-7. As polar lipids, phosphatidylethanolamine, two aminophospholipids, two aminolipids, a glycolipid and three unknown lipids were detected on the TLC plate. The DNA G+C content of strain EX36T was 47.2 mol%, which is lower than reported values for all other species of the genus Spirosoma with validly published names.The predominant menaquinone, in accordance with all other species of the genus T with its nearest phylogenetic neighbour S. linguale DSM 74T was also carried out by the Identification Service of the DSMZ. The experiment was performed in duplicates. DNA−DNA hybridization showed a DNA−DNA similarity of 12.2 % (second measurement: 17.2 %), demonstrating that these two strains do not represent the same species.The analysis of DNA−DNA similarity of strain EX36T represents a distinct species in the genus Spirosoma, for which the name Spirosomaendophyticum sp. nov. is proposed.The present data regarding 16S rRNA gene sequence analysis, physiological, chemotaxonomic and morphological properties indicates, that strain EX36Spirosom endophyticum .N-acetylglucosamine, gluconate, caprate, adipate, malate, citrate, phenylacetate, β-methyl d-glucoside, d-salicin, n-acetyl-β-d-mannosamine, n-acetyl neuraminic acid, d-galactose, d-fucose, l-fucose, l-rhamnose, inosine, d-arabitol, myo-inositol, d-aspartic acid, d-serine, glycyl-l-proline, l-alanine, l-arginine, l-aspartic acid, l-glutamic acid, l-serine and pectin. The following substrates are weakly utilized: dextrin, maltose, trehalose, cellobiose, gentiobiose, sucrose, turanose, stachyose, α-lactose, melibiose, α-d-glucose, d-mannose, d-fructose, d-mannitol and l-histidine. d-Raffinose and N-acetyl-d-glucosamine are utilized. Susceptible to the following antibiotics (µg per disc): streptomycin (10), kanamycin (30), chloramphenicol (60) and rifampicin (15) and resistant to ampicillin (10), polymyxin B (20), tetracycline (15) and erythromycin (15). The major fatty acids are summed feature 3 (iso-C15 : 0 2-OH and/or C16 : 1ω7c), C16 : 1ω5c, iso-C17 : 0 3-OH and iso-C15 : 0; the complete fatty acid profile can be found in Cells are rod-shaped, Gram-reaction-negative, non-spore-forming, with a size of 1.2×2–17.5 µm. A yellow pigment which is not of the flexirubin type is produced. Filaments up to 55 µm may be formed. Colonies on 10 % TSA are opaque, semi-translucent with a smooth and shiny surface and a circular, convex shape. Aerobic growth occurs at 20–28 °C (optimum at 28 °C), pH 5–8 (optimum at pH 7); tolerates concentrations up to 0.6 % NaCl (w/v) in the medium, whereas best growth was achieved in absence of NaCl. Positive for catalase and oxidase activity. Nitrate is not reduced and indole is not produced. Negative for glucose fermentation, hydrolysis of arginine and gelatin, and urease activities and positive for aesculin hydrolysis. Does not utilize the following substrates: arabinose, mannitol, T ( = DSM 26130T = LMG 27272T), was isolated from Zn/Cd-accumulating Salix caprea in Arnoldstein, Austria. The DNA G+C content of the type strain is 47.2 mol%.The type strain, EX36


We therefore investigated whether the elevated maltose level in mex1-1 in the warm could result in changes in metabolism and physiology typical of WT plants grown in the cold.Arabidopsis plants accumulate maltose from starch breakdown during cold acclimation. The Arabidopsis mutant, mex1-1 plants were much smaller, with fewer leaves, and elevated carbohydrates and amino acids compared to WT. However, after transfer to 4 °C the total soluble sugar pool and amino acid concentration was in equal abundance in both genotypes, although the most abundant sugar in mex1-1 was still maltose whereas sucrose was in greatest abundance in WT. The chlorophyll a/b ratio in WT was much lower in the cold than in the warm, but in mex1-1 it was low in both warm and cold. After prolonged growth at 4 °C, the shoot biomass, rosette diameter and number of leaves at bolting were similar in mex1-1 and WT.Grown at 21 °C, mex1-1 mutation in warm-grown plants confers aspects of cold acclimation, including elevated levels of sugars and amino acids and low chlorophyll a/b ratio. This may in turn compromise growth of mex1-1 in the warm relative to WT. We suggest that elevated maltose in the plastid could be responsible for key aspects of cold acclimation.The  To survive in temperate climates, cold-hardy plants such as Arabidopsis make rapid genetic and metabolic changes in response to a reduction in temperature , 2]. Ch. Ch2]. CBAM3 (BMY8), which is essential for maltose production, and GLUCAN WATER DIKINASE1 (SEX1), which mediates the phosphorylation of amylopectin. In both cases the mutants exhibited a freezing-sensitive phenotype, providing evidence that maltose accumulation is important for cold acclimation [Several genes involved in starch metabolism have been observed to increase in expression in response to rapid cooling  8] 9]. [9].8] [. [9].8] limation , 12]. . BAM3 (B2) cycle , , , 11]. F. F11]. F2) cycle . Accumul2) cycle , 17]. C. C2) cyc2) cycle . MALTOSE EXCESS1 (MEX1) gene encodes a maltose transporter that is localized to the chloroplast envelope and is required for maltose export into the cytosol . H. HMALTOSArabidopsis thaliana mex1-1 mutant seeds were kindly donated by S. C. Zeeman (ETH Zurich). Wild type Arabidopsis L. ecotype Columbia (Col-0), and mex1-1 mutant seeds were sown on 0.25 L pots of compost. Seeds were stratified for 3 days at 4 °C and transferred to growth chambers  in a 12 h light:12 h dark photoperiod. For metabolite and carbohydrate analyses, plants were grown on compost as above for 34 days before transfer to 4°C in a 12 h light:12 h dark photoperiod. For morphological and leaf pigmentation phenotyping, plants were germinated as above and then maintained at 21 °C or transferred to 4°C after 14 days. For phenotyping, plants were also maintained in their respective growth conditions until bolting.13C6 and L-valine-13C5,15N valine, and 5 µg/ml each of 2-aminoanthracene and pentafluorobenzoic acid. The sample mixtures were vortexed for 30 s and incubated at 70°C for 15 min. Milli-Q Water (500 µL) was added to the extracts, vortexed for a further 30 s and then centrifuged for 15 min at 13,000 rpm at room temperature  to pellet cellular debris. Aliquots  from the sample extracts were dried in vacuo for subsequent derivatisation and analysis by GC-MS or LC-MS. Plants were grown at 21°C for 34 days before transfer to 4°C for 5 days. Whole rosettes were harvested 11 h into the 12 h light period on these days, and immediately frozen in liquid nitrogen. Approximately 60 mg fresh weight (FW) ground samples were weighed into pre-chilled 2 mL microfuge tubes. Ground samples were extracted in 500 µL of 100% methanol that contained 10 µg/ml each of the internal standards D-sorbitol--1 methoxyamine hydrochloride in pyridine and derivatised at 37°C for 120 min with mixing at 500 rpm. The samples were then treated for 30 min with 20 µL N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) and 2.5 µL retention time standard mixture  with mixing at 500 rpm. The derivatised samples were allowed to rest for 60 minutes prior to injection. Samples (1 µL) were injected using a hot needle technique into a GC-MS system comprised of a Gerstel 2.5.2 autosampler, a 7890A Agilent gas chromatograph and a 5975C Agilent quadrupole MS . The MS was adjusted according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). The GC was performed on a 30 m VF-5MS column with 0.2 µm film thickness and a 10 m Integra guard column . The injection temperature was set at 250°C, the MS transfer line at 280°C, the ion source adjusted to 250°C and the quadrupole at 150°C. Helium was used as the carrier gas at a flow rate of 0.8 mL min-1. The analysis of samples was performed under the following temperature program; start at injection 70°C, a hold for 1 min, followed by a 7°C min-1 oven temperature, ramp to 325°C and a final 6 min heating at 325°C.GC-MS was done based on Roessner et al. (2001) . The dri-1 with an m/z 50-600 scanning range. Both chromatograms and mass spectra were evaluated using the Chemstation program . Mass spectra of eluting compounds were identified using the commercial mass spectra library NIST 05 (http://www.nist.gov), the public domain mass spectra library of Max-Planck-Institute for Plant Physiology, Golm, Germany (http://csbdb.mpimp-golm.mpg.de/csbdb/dbma/msri.html) and the in-house Metabolomics Australia mass spectral library. All matching mass spectra were quantified using by determination of the retention time and authentic sugar calibration standards.Mass spectra were recorded at 2 scans sLC-MS was done essentially as described in Boughton et al. (2011) , modifie-1, nebulizer pressure 45 psi and capillary voltage 3500 V. An Agilent Zorbax Eclipse RRHD-C18 Rapid Resolution HT 2.1 x 100 mm, 1.8 µm column was used with a flow rate of 800 µL min-1, maintained at 40°C, resulting in operating pressures below 1000 bar with a 10 min run time. "A gradient LC method was used with mobile phases comprised of (A) water/ 0.1% formic acid and (B) acetonitrile/ 0.1% formic acid. The proportion of B in the gradient and the times (min) were: 1 % B (0 - 0.5 min), 1-10% B (0.5 - 3.5 min), 10-20% B (3.5 - 6.0 min), 20-100% B (6.0 - 6.1 min), 100% B (6.1 - 7.0 min), 100-1% B (7.0 - 7.1 min), 1% B (7.1 - 10.0 min).For derivatisation, 10 µL aliquots of each standard or sample was added to 70 µL of borate buffer  containing 10 mM TCEP, 1 mM ascorbic acid and 50 µM 2-aminobutyric acid. The resulting solution was vortexed, then 20 µL of AQC reagent (200 mM dissolved in 100% ACN) was added and immediately vortexed. The samples were heated with shaking at 55°C for 10 minutes then centrifuged and transferred to HPLC vials containing inserts. Derivatized samples were quantitatively analysed using an Agilent 1290 Infinity LC-system coupled to an Agilent 6490 Electrospray Ionisation-Triple Quadrupole MS. Injection volumes of 1 µL of samples or standards were used. Ions were monitored in the positive mode using a MRM method optimized for each analyte. The source, collision energies and fragmentor voltages were optimized for each analyte by infusing a derivatised standard with LC eluent. The following source conditions were used: sheath gas temperature 400°C, gas flow 11 L minThese conditions provided suitable chromatographic separation of modified amino acids and although co-elution was observed for some of the species, this could be overcome by the mass-selective capabilities of the mass spectrometer using MRM. Agilent MassHunter Quantitative Analysis Software, Version 6.0 was used to quantify levels of amino acids. mex1-1 and WT were harvested from plants kept at 21°C for 4 weeks  or after transfer to 4°C for ~131 days . Five biological replicates were harvested per genotype, per treatment. Samples were weighed and then frozen in liquid nitrogen. Chlorophyll was quantified as described by a and b, respectively. Total chlorophyll a and b was determined using the following formulae: (12.7 x A663 - 2.69 x A646) x Volume / Weight = Chl a mg/g FW and (22.9 x A646 - 4.86 x A663) x Volume / Weight = Chl b mg/g FW.Two fully expanded rosette leaves of Samples were harvested, weighed and ground as for chlorophyll. Anthocyanins were extracted and quantified according to : Ground (Abs 530 – Abs 630) / weight = anthocyanin units /g FW, where one unit of anthocyanin equals one absorbance unit in 1 ml of extraction solution.Statistical analyses were performed using GenStat 13th Edition  and Microsoft Excel. For metabolites, a two way ANOVA and accompanying Tukey test was performed to identify significant differences between genotypes and treatments. For carbohydrates, leaf pigments and morphology, Student’s two-tailed T-tests assuming unequal variances were performed. Significance for both tests was set at 0.05.mex1-1 in warm and cold conditions, respectively. In both treatments GABA was in lowest abundance in mex1-1 and also in WT in the warm, after acclimation Ornithine was of lowest abundance in the WT  cycle in which glycine, produced in peroxisomes, is converted within the mitochondria to Ser . F. Fmex1-1WT plant . mex1-1 are similar to those reported for the Arabidopsis eskimo1 mutant that, unlike WT plants, exhibits constitutive freezing tolerance without requiring a period of acclimation . H. Hmex1-1te as WT . In thesmex1-1 to accumulate high levels of Gly in response to cold acclimation suggests that mex1-1 is deficient in this aspect of cold acclimation. As cold acclimation is part of the adaptive process that prepares the plant to withstand freezing temperatures, it is feasible that this deficiency may confer greater susceptibility to freezing stress. As a freezing sensitive phenotype has been reported for the starch metabolism mutants β–amylase3/bmy9 and sex1 it would be interesting to determine whether they are also deficient in the accumulation of Gly during cold acclimation . T. T11]. Tlimation , 8]..mex1-1 tmex1-1, it is feasible that this stimulates greater uptake and reduction of nitrates resulting in the increased abundance of N storing amino acids, such as Gln and Glu, as observed under warm conditions. However, as mex1-1 is dwarf under warm conditions it is clearly unable to capitalize on its high nutrient status for growth, possibly owing to carbon-limitation at night , . I. Imex1-1mex1-1 mutant plants grown at 21°C, the mutant shows greater similarity to WT, in terms of biomass, amino acids, total carbohydrates and chlorophyll when grown in the cold. This is probably the result in mex1-1 of a pre-existing shift away from plastidial starch-derived carbon to cytosolic carbon reserves that mirrors changes occurring in plants undergoing cold acclimation. The results further suggest that maltose accumulation could, in part, mediate some of the effects of chilling on plant growth, including changes to chlorophyll a/b ratio and amounts of amino acids and sugars. In summary we have discovered that despite the differences between wild type and


Halobacterium salinarum is dependent upon Mn-antioxidant complexes responsible for the scavenging of reactive oxygen species (ROS) generated by radiation. Here we examined the role of the compatible solutes trehalose, mannosylglycerate, and di-myo-inositol phosphate in the radiation resistance of aerobic and anaerobic thermophiles. We found that the IR resistance of the thermophilic bacteria Rubrobacter xylanophilus and Rubrobacter radiotolerans was highly correlated to the accumulation of high intracellular concentration of trehalose in association with Mn, supporting the model of Mn2+-dependent ROS scavenging in the aerobes. In contrast, the hyperthermophilic archaea Thermococcus gammatolerans and Pyrococcus furiosus did not contain significant amounts of intracellular Mn, and we found no significant antioxidant activity from mannosylglycerate and di-myo-inositol phosphate in vitro. We therefore propose that the low levels of IR-generated ROS under anaerobic conditions combined with highly constitutively expressed detoxification systems in these anaerobes are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.Radiation-resistant bacteria have garnered a great deal of attention from scientists seeking to expose the mechanisms underlying their incredible survival abilities. Recent analyses showed that the resistance to ionizing radiation (IR) in the archaeon  The vast majority of cellular insults under aqueous conditions are caused by indirect effects, through the action of reactive oxygen species (ROS) formed by the radiolysis of water and generating hydroxyl radicals (HOe H2O2) . DNA-asse (H2O2) , 2. ProtO2 . DNA-e (H2O2) , 4. Protreaction . PreventWhile it was thought that DNA lesions, and in particular DNA double-strand breaks (DSBs), were the most cytotoxic lesions resulting from IR exposure, recent findings regarding the repair of DNA from IR damage and the fact that IR-sensitive and IR-resistant organisms suffer the same number of DNA DSBs for an equivalent dose of IR (~0.01 DSB/Gy/Mbp) strongly departed from this dogma , 7. It iHalobacterium salinarum revealed the critical role played by nonenzymatic antioxidant processes in the radioresistance of this organism [H. salinarum, in addition to being adapted to high salt, also exhibits high-level resistance to desiccation, high pressure, UV radiation, and IR [D10, the dose of radiation in Gray (Gy) that reduces the survival of a population by 90%, is 5 kGy [H. salinarum cell interior revealed a high manganese/iron (Mn/Fe) ratio similar to that of the extremely radiation-resistant bacterium Deinococcus radiodurans (D10 12 kGy) and other IR resistant microorganisms [D. radiodurans elegantly established the key role played by Mn2+-peptide complexes in this bacterium's radiation resistance [in vivo studies showed the important function of Mn-orthophosphate complexes in oxidative stress [Bacillus, Mn2+-dipicolinic complexes are implicated in the stress resistance phenotypes of spores, including IR, wet and dry heat [2+ and mycosporine-like amino acids [H. salinarum enzyme-free cell extracts rich in Mn, phosphate, amino acids, and peptides provided a high level of enzyme protection, in vitro, against the deleterious effect of IR, underlying that the critical role of Mn antioxidant complexes in radiation resistance also extends to archaea [Regarding the mechanisms underlying IR resistance, recent studies with the halophilic archaeon organism , 11. H. , and IR . Its D1is 5 kGy . Measuresistance  and in ye stress . In Bacidry heat , and cyano acids . In H. s archaea . D. radiodurans and environmental isolates, a strong link was established between desiccation and IR resistance [Ustilago maydis [Philodina roseola [ Milnesium tardigradum [Caenorhabditis elegans [Archaeoglobus fulgidus, methanogens such as Methanocaldococcus jannaschii, the hyperthermophiles P. furiosus, Thermococcus radiotolerans, and Thermococcus gammatolerans [Rubrobacter xylanophilus and Rubrobacter radiotolerans [Many extremophiles have been found to be resistant to IR, suggesting that radiation resistance is a fortuitous consequence of a high tolerance to other environmental stressors . From stsistance , 22. Botsistance . Howeversistance , 24. Theo maydis , the fre roseola , the watdigradum , and the elegans . A numbetolerans , 29–31, tolerans , 33. Howtolerans .in vitro [myo-inositol phosphate (DIP), a compatible solute exclusively found in thermophiles, is accumulated in the cell in response to thermal stress [in vitro against thermal stress and freeze drying [Thermophilic bacteria and archaea inhabit diverse environments and can survive multiple stresses including desiccation, radiation, pressure, and pH extremes together with high temperature , 34, 35.in vitro , 40. Manin vitro . Di-myo-l stress , 41. Bote drying –45. R. xylanophilus (D10 6 kGy) and R. radiotolerans (D10 10 kGy), and two IR-resistant hyperthermophilic archaea, P. furiosus (D10 3 kGy) and T. gammatolerans (D10 6 kGy). We showed that under aerobic conditions, compatible solutes accumulated by thermophilic bacteria confer IR resistance to enzymes in vitro and that radioprotection is mitigated by the presence of both trehalose and  Mn2+. With regard to hyperthermophilic archaea, the anaerobic environment contributes to their IR resistance, which was the most significant factor for protection of enzymes in vitro. In this study, we investigated the role played by the compatible solutes found in two IR-resistant thermophilic bacteria,  radiotolerans (DSM 5868) and Thermococcus gammatolerans (DSM 15229) were obtained from the DSMZ. Rubrobacter xylanophilus (DSMZ 9941) was a gift from Dr. Gaidamakova. Rubrobacter spp. were grown in TM medium containing 1 g/L tryptone, 1 g/L yeast extract, 0.7 g/L NaNO3, 0.1 g/L Na2HPO4, 0.1 g/L nitrilotriacetic acid, 0.1 g/L MgSO4·7H2O, 0.1 g/L KNO3, 60 mg/L CaSO4·2H2O, 8 mg/L NaCl, 2.2 mg/L MnSO4·H2O, 0.5 mg/L ZnSO4·7H2O, 0.5 mg/L H3BO3, 25 μg/L CuSO4·5H2O,  25 μg/L Na2MoO4·2H2O, 46 μg/L CoCl2·6H2O, 10 ml/L 0.17 mM FeCl3·6H2O, final pH 8.2. Cultures were grown at 48°C for R. radiotolerans and at 60°C for R. xylanophilus, with shaking at 220 rpm in a Gyromax 737 shaker . Pyrococcus furiosus strain (DSMZ 3638) was grown in the absence of sulfur with 100 μM Na2WO4 and 0.5% (wt/vol) maltose in the Pf medium containing 24 g/L NaCl, 4 g/L Na2SO4, 0.7 g/L KCl, 0.2 g/L NaHCO3, 0.1 g/L KBr, 0.03 g/L H3BO3, 10.8 g/L MgCl2·6H2O, 1.5/Lg CaCl2·2H2O, 0.025 g/L SrCl2·6H2O, 0.08% Na2S·9H2O, 5 g/L tryptone, 1 g/L yeast extract, 1 ml/L resazurin (0.2 g L−1 solution), final pH 6.8, in 100 mL serum bottles or 1 L bottles at 95°C under anaerobic conditions [Thermococcus gammatolerans was grown in ASW-YTP medium containing 38 g/L NaCl, 14.5 g/L MgCl2·6H2O, 5 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium pyruvate, 5.6 g/L MgSO4·7H2O, 2.5 g/L CaCl2·2H2O, 2.6 g/L Na2SO4, 1 g/L KCl, 80 mg/L Na2CO3, 80 mg/L NaBr, 64 mg/L KBr, 58 mg/L SrCl2·6H2O, 42 g/L H3BO3, 8.1 mg/L Na2HPO4, 2.4 mg/L NaF, 0.4 mg/L NaSiO4, 50 μg/L KI, 0.08% Na2S·9H2O, 1 ml/L resazurin (0.2 g L−1 solution), final pH 6.8, in 100 mL serum bottles or 1 L bottles under anaerobic conditions at 88°C. Rubrobacter600 nm  and the cells harvested by centrifugation at 8,000 ×g . Rubrobacter spp. cells were washed twice with TM-BSS , P. furiosus cells with Pf-BSS , and T. gammatolerans cells with ASW-BSS . Pellets were resuspended in distilled and deionized water  and passed through an Emulsiflex Homogenizer  at 15,000 psi to lyse the cells. Cell extracts were centrifuged at 12,000 ×g  and standardized by protein concentration, which was determined by the BioRad Bradford Assay . The supernatant was further centrifuged at 190,000 ×g  and subjected to filtration using 3 kDa centrifugal devices . The resulting protein-free cell extracts, called ultrafiltrates (UFs), were concentrated 5 times in a speed vacuum concentrator  and stored at −20°C. The UF for H. salinarum was prepared as described in [Cultures were grown in appropriate media and conditions to 0.4  ODribed in .DdeI was added at a final concentration of 0.5 unit/μL to UFs diluted to 0.2x, to 25 mM phosphate buffer (PiB), pH 7.0, to a 20 mM solution of trehalose, mannosylglycerate (MG), or di-myo-inositol phosphate (DIP), with or without the addition of 250 μM or 25 μM MnCl2. Assays performed under anaerobic conditions were purged with ultrahigh purity Ar (99.999%) . The solutions were irradiated on ice using a 60Co gamma source  at the following doses: 0, 1, 2, 3, 4, and 5 kGy or 0, 1, 2, 4, 6, 8, 10, and 12 kGy. Samples were kept on ice until digestion of 1 μg of pUC19 DNA using 1 U of enzyme from each irradiated solution at 37°C for 1 h. The resulting pUC19 DNA fragments were separated by electrophoresis on 1% agarose TBE gels and visualized with ethidium bromide staining as previously described [The restriction enzyme escribed .R. xylanophilus, R. radiotolerans, P. furiosus, and T. gammatolerans were determined using the ninhydrin assay as previously described [Free and total amino acid concentrations in the UFs of escribed . 4 concentration in R. xylanophilus, R. radiotolerans, P. furiosus, and T. gammatolerans UFs and cells  were determined using ICP-MS  and ion chromatography (PO4) at the Division of Environmental Health Engineering, JHU School of Public Health as previously described [Mn, Fe, and POescribed . 9 cells were resuspended in 80% ethanol, broken via French press as previously described [2O, Sigma-Aldrich) before filtration through a 10 kDa filter . Cell protein concentration was determined by the BioRad Bradford Assay  using cell pellets of 109 cells resuspended in distilled and deionized water , lysed by French press, and centrifuged as described above. Cells were harvested and washed with BSS. Pellets of 10escribed , and cenmyo-inositol phosphate (DIP) were obtained from Bitop AG, Witten, Germany. High-performance anion-exchange chromatography (HPAEC) was carried out on Dionex DX 500 with a CarboPac PA-10 column and a PA-10 guard column  and pulsed amperometric detection (PAD). An aliquot of the ethanol extract was diluted 10- to 100-fold and injected into a CarboPac PA-1 column equilibrated with 16 mM sodium hydroxide. Elution was performed with a linear gradient from 16 mM sodium hydroxide to 0.5 M sodium acetate/0.1 M sodium hydroxide. Standards of 0.25, 0.5, 1, 2, and 4 nmol of trehalose, MG, and DIP were run for quantification. Mannosylglycerate (MG) and di-2+ and small organic molecules that included amino acids and peptides [ in vitro at physiologically relevant concentrations, these constituents formed potent antioxidant complexes in orthophosphate buffer (PiB) [R. xylanophilus, R. radiotolerans, P. furiosus, and T. gammatolerans , of IR-resistant bacteria and archaea were found enriched in Mnpeptides , 15. Wheer (PiB) , 15. To alinarum . The con. putida . The Mn/. putida . Phosphafuriosus .R. radiotolerans and R. xylanophilus UFs both contained high amounts of trehalose with 29 mM and 17 mM, respectively. In addition, we found mannosylglycerate (MG) in UFs of both R. xylanophilus (99 mM) and R. radiotolerans (64 mM), whereas only the R. xylanophilus UF contained di-myo-inositol phosphate (DIP) (33 mM)  to quantify compatible solutes in UFs.  (33 mM) . P. furintration . Thus, tP. furiosus were similar to previously reported concentration of MG obtained by NMR, validating our methodology. Using cells grown in similar growth conditions with respect to salinity and temperature, we calculated an intracellular concentration of MG of 0.22 μmol/mg protein versus 0.25 μmol/mg protein reported by Martin and Santos [P. furiosus and T. gammatolerans, using a cellular volume of 4.5 μL/mg protein [P. furiosus, MG and DIP concentrations were 49 mM and 10 mM, respectively, and in T. gammatolerans, we found concentrations of 21 mM for MG and 11 mM for DIP. These intracellular concentrations were similar to those for MG and DIP in the UFs of those organisms  than P. furiosus and T. gammatolerans in both the UFs and in our whole cells (ethanol extracts) determinations. To estimate intracellular concentrations of compatible solutes, we analyzed the ethanol extracts of our thermophilic organisms using HPAEC . Our datd Santos . We calc protein . In P. frganisms . With reTo investigate the role of those small molecules in radiation resistance, we tested the ability of UFs and of reconstituted preparations, to protect the activity of purified enzymes exposed to increasing doses of IR.R. xylanophilus and R. radiotolerans on the activity of DdeI, a restriction endonuclease, exposed to doses of IR up to 12 kGy  protected enzyme activity of 2 kGy, the addition of trehalose resulted in a significant increase in protection, up to 6 kGy , the radioprotection increased dramatically to 12 kGy. Irradiating the enzyme in PiB and Mn2+ alone only protected its activity to 2 kGy, and the addition of 25 mM MG or DIP did not increase protection , we tested the UFs' properties under anaerobic conditions. In the absence of O2, UFs of P. furiosus and T. gammatolerans protected DdeI up to 3 kGy , and R. radiotolerans is considered moderately thermophilic with an optimal growth temperature of 48°C [R. xylanophilus, in contrast, has an optimal growth temperature of 60°C, which is the lowest growth temperature reported among organisms known to accumulate DIP [R. xylanophilus accumulates trehalose, MG, and DIP under its optimal growth conditions and increases the concentration of these compatible solutes in response to heat or osmotic stress [R. radiotolerans also maintains basal millimolar cellular concentrations of trehalose and MG, representing organic solutes constitutively present in the cell, with potential for antioxidant properties. While it was unexpected that MG and DIP did not protect enzyme activity in vitro, these compatible solutes carry negative charges and might be repelled at various negatively charged sites on a protein, leaving areas susceptible to ROS attacks. Although, both compounds were previously shown to scavenge HO• [2•− and H2O2, generated by exposure to IR [Compatible solutes of thermophiles have been studied extensively for their protein-stabilizing properties . A numbe of 48°C . R. xylalate DIP . As prevc stress . We haveenge HO• , we concre to IR .2+. Trehalose is present in a wide variety of organisms including bacteria, yeast, fungi, plants, and invertebrates and was found to protect proteins from heat, osmotic stress, desiccation, and oxidation [Chroococcidiopsis, a desiccation and IR-resistant cyanobacterium, were shown to accumulate trehalose [R. xylanophilus and R. radiotolerans. These findings fit the current model of Mn-based antioxidants scavenging IR-generated ROS that was established for aerobic bacteria and archaea [Trehalose, a compatible solute of similar size as MG and DIP but carrying no charges, was highly protective of protein activity against IR, either alone or in combination with Mnxidation . Additiorehalose , 56. Her archaea , 11, 51. archaea .P. furiosus and T. gammatolerans seems to be quite different from that of the aerobic thermophiles. Both organisms exhibited low Mn/Fe ratios that were similar to those of radiation-sensitive E. coli and P. putida [2+-dependent ROS scavenging for aerobic bacteria and archaea [P. furiosus uses in place of NAD [P. furiosus, like most anaerobic hyperthermophiles, lacks the oxygen detoxification enzymes superoxide dismutase (SOD) and catalase that are used by their aerobic counterparts [P. furiosus has a superoxide reductase (SOR), a nonheme iron-containing enzyme that catalyzes the reduction of O2•− into H2O2, and several peroxidases, including rubrerythrin, and alkyl hydroperoxide reductase I and II [2+-dependent ROS scavenging of O2•− and H2O2—which are formed predominantly under aerobic irradiation—is essential in the presence of oxygen, it might not be the case under anaerobic conditions [2, the formation of O2•− is a 1-step process in which a free electron (e−) reacts with O2 (2.0 1010 M−1 s−1). This is significantly faster than in the absence of O2 where the formation of O2•− is dependent upon concentrations of HO• and H2O2 (2.7 107 M−1 s−1) [in vitro protection assay, P. furiosus and T. gammatolerans UFs displayed increased protection under anaerobic conditions, suggesting that one element of their radiation resistance might be attributed to the anaerobic environment itself. Another element is likely to be their metabolic adaptations to maintaining strict anaerobic conditions in their intracellular milieu. The basis for the radiation resistance of the anaerobic hyperthermophiles . putida . This is archaea , 11, 51.e of NAD –60. P. fterparts . InsteadI and II , 62. Furnditions , 64. In M−1 s−1) . In our H. salinarum to IR [P. furiosus in response to radiation showed that genes encoding for a putative Dps-like iron-chelating protein and two membrane-bound oxidoreductases were differentially expressed following IR, potentially in response to oxidative stress [P. furiosus under normal anaerobic growth conditions, and no increased expression of SOR was found in response to IR or H2O2, indicating that this protein may be functioning at maximum capacity at all times [2•− detoxification was recently discovered in Desulfoarculus baarsii in which SOR complexed with ferrocyanide reduces O2•− without the formation of H2O2. This system is highly efficient, as the SOR iron site remains reduced, thus eliminating the requirement of oxidoreductases to recycle SOR [P. furiosus and T. gammatolerans, are key to their radiation resistance and circumvent the need for the accumulation of Mn-antioxidant complexes in the cell.While ROS detoxification enzymes were shown to be dispensable for the survival of the aerobic archaeon um to IR , a wholee stress . The mose stress . Similarll times , 62, 65.ycle SOR . We prop2+-dependent ROS scavenging extend to eukaryotic systems? The study of extremophiles and how they meet the physical and chemical challenges found in the environmental extremes they inhabit has led to many new insights into the mechanisms of stress response. Previous work, together with the mechanisms underlying the radiation resistance of the thermophiles described here, underscores the multiple strategies microorganisms can use to escape environmental stresses. The variety of Mn-antioxidant complexes found so far suggests that the adaptations of extremophiles to their environments provide a tremendous reservoir for novel radioprotective molecules and antioxidants against the deleterious effects of IR. One question remains: is Mn a universal feature of IR resistance in aerobic systems, and does the model of Mn


Background: Ki-67 is a robust predictive/prognostic marker in prostate cancer; however, tumor heterogeneity in prostate biopsy samples is not well studied. Methods: Using an MRI/US fusion device, biopsy cores were obtained systematically and by targeting when indicated by MRI. Prostate cores containing cancer from 77 consecutive men were analyzed. The highest Ki-67 was used to determine interprostatic variation. Ki-67 range (highest minus lowest) was used to determine intraprostatic and intralesion variation. Apparent diffusion coefficient (ADC) values were evaluated in relation to Ki-67. Results: Interprostatic Ki-67 mean ± standard deviation (SD) values for NCCN low (L), intermediate (I), and high (H) risk patients were 5.1 ± 3.8%, 7.4 ± 6.8%, and 12.0 ± 12.4% (ANOVA P = 0.013). Intraprostatic mean ± SD Ki-67 ranges in L, I, and H risk patients were 2.6 ± 3.6%, 5.3 ± 6.8%, and 10.9 ± 12.3% (ANOVA P = 0.027). Intralesion mean ± SD Ki-67 ranges in L, I, and H risk patients were 1.1 ± 0.9%, 5.2 ± 7.9%, and 8.1 ± 10.8% (ANOVA P = 0.22). ADC values at Ki-67 > and <7.1% were 860 ± 203 and 1036 ± 217, respectively (P = 0.0029). Conclusions: High risk patients have significantly higher inter- and intraprostatic Ki-67 heterogeneity. This needs to be considered when utilizing Ki-67 clinically. Progress in multiparametric MRI imaging has improved our ability to visualize specific target lesions within the prostate. Ultrasound/MRI fusion devices allow for targeted biopsies of these specific MRI defined lesions. These advances create an opportunity to evaluate biomarkers from specific target lesions for integration into radiation treatment stratification.The Ki-67 protein functions as a nuclear antigen that is only expressed in proliferating cells. It is a marker of the growth fraction in malignant tissue –3. It isThis was an IRB approved retrospective study. Charts were reviewed for patients who were referred to the Department of Urology for Artemis (ultrasound/MRI fusion) guided prostate biopsies. All men underwent 3T multiparametric MRI prior to biopsy. Lesions identified on MRI imaging were segmented as regions of interest. The MRI was then fused with ultrasound at the time of the biopsy. Systematic Artemis assisted biopsies were performed first and, when MRI indicated a lesion, targeted biopsies were performed. Targeted biopsies were taken every 3–5 mm through a target.Patients were stratified by NCCN Risk criteria using pretreatment PSA, T stage, and Gleason score. Pathology reports were reviewed for Gleason score and Ki-67 (%) for each of the positive prostate cancer cores. The highest Ki-67 documented for each patient was used for interprostatic variation. For patients with ≥2 positive biopsies variation within each prostate (intraprostatic) was performed by taking the highest Ki-67 minus the lowest Ki-67. Intralesion analysis was carried out when multiple biopsy cores were taken from one MRI-defined lesion using the same high minus low Ki-67 method used for intraprostatic variation. The index lesion was defined as the one with the maximum tumor diameter as measured on T2 weighted MRI. The ADC values of lesions as determined from diffusion weighted imaging were also examined to determine if there was a correlation with Ki-67.μm thickness and paraffin removed with xylene and rehydrated through graded ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval (HIER) was carried out for all sections in 0.01 M Citrate buffer, pH = 6.00, using a vegetable steamer at 95°C for 25 min. The slides were then stained with mouse monoclonal Ki-67, clone MIB1 , for 45 min at room temperature. The primary antibody was diluted with calcium chloride to 1/100 concentration. The signal was detected using the MACH 2 Mouse HRP Polymer . All sections were visualized with the diaminobenzidine reaction and counterstained with hematoxylin.Paraffin-embedded sections were cut at 4 P  value < 0.05.One-way analysis of variance (ANOVA) was used to evaluate significant differences between the means of the different NCCN Risk groups. Statistical significance was set at a 77 men were identified who had Artemis guided positive prostate biopsies with Ki-67 staining reported. The mean patient age was 67, the mean PSA was 7.7 ng/dL, and all patients had a clinical stage of T2a or lower see .P = 0.013)  values for low, intermediate, and high risk patients of 5.1% ± 3.8%, 7.4% ± 6.8%, and 12.0% ± 12.4% (ANOVA = 0.013) . It was P = 0.027). Intraprostatic variation was assessed on 47 patients with ≥2 biopsy-positive cores with Ki-67 quantified. Mean ± SD Ki-67 variation (the highest Ki-67 minus the lowest Ki-67) in low, intermediate, and high risk patients was 2.6 ± 3.6%, 5.3 ± 6.8%, and 10.9 ± 12.3% (ANOVA P = 0.22).Intralesion variation was assessed on 38 MP-MRI defined lesions that had ≥2 cores from each lesion with Ki-67 staining. Intralesion mean ± SD Ki-67 variation (the highest Ki-67 minus the lowest Ki-67) in low, intermediate, and high risk patients was 1.1 ± 0.9%, 5.2 ± 7.9%, and 8.1 ± 10.8% (ANOVA 10 patients had 2 or more lesions identified on MP-MRI. The dominant lesion harbored the highest Ki-67 30% of the time . The domn = 31) was 1075 ± 205 and in patients with a Ki-67 > 3.5% (n = 48) was 940 ± 224 (P = 0.0039). For patients with a Ki-67 < 7.1% (n = 60) the mean ADC was 1036 ± 217 while patients with values > 7.1% had a mean ADC of 860 ± 203 (P = 0.0029).Ki-67 cut-off levels of <3.5% and >7.1% were used based on retrospective validation of these values in predicting outcomes following definitive radiation treatment in patients treated on two separate RTOG trials , 16. TheNCCN risk grouping  is commonly used to determine radiation treatment options. While this risk stratification is clinically helpful, it is also limited in that patients in each risk category are not homogeneous. Integration of biomarkers into existing stratification schemes could help personalize treatment options. Ki-67 is a robust biomarker that has been evaluated in three separate RTOG trials with cut-offs of 3.5%, 6.2%, and 7.1% being independent predictors of outcomes , 15–17. Using information from multiparametric MRI and targeted biopsies we were able to demonstrate a number of things. Overall we found Ki-67 levels are more heterogeneous with increasing NCCN risk group. This observation is consistent with other studies which have shown increased Ki-67 in patients with higher Gleason scores , 19. We Given the variability within the prostate we tried to determine if the index lesion was most likely to harbor the highest Ki-67. We found that this was the case in only 3/10 cases. This suggests that relying on a biopsy only from the index lesion may not be a reliable representation of the highest Ki-67 within the entire gland.Further complicating things is the high variability within an individual lesion. While 8% variability (the highest Ki-67 minus lowest Ki-67) seems low the cut-offs for Ki-67 levels that stratify patients range between 3.5% and 7.1%. So a difference of 8% is actually very meaningful. The concept of tumor heterogeneity is certainly not novel but this study emphasizes the importance of not relying too heavily on a single core. A more representative picture of the tumor, at least with respect to Ki-67, is better achieved with multiple cores taken from a single lesion. It is beyond the scope of this paper to provide an answer to how many cores are needed to accurately depict the totality of tumor heterogeneity.Given the complexities with accurately portraying tumor heterogeneity based off of biopsy samples we asked whether the average ADC value correlates with Ki-67 values. This would be meaningful because it would be much simpler to use an ADC value generated from a computer to stratify a patient rather than doing multiple targeted biopsies. We found that there was a significant correlation between ADC values within clinically relevant Ki-67 groupings . There is overlap between these values but their means are significantly different from one another. While this is an interesting correlation, validation on a larger cohort is needed and ultimately prospective data is needed to determine if ADC values can independently predict outcomes in prostate cancer.This study provides the first evidence of the magnitude of tumor heterogeneity of the most well studied tumor biomarker in radiation therapy for prostate cancer. Integration of Ki-67 into future risk stratification schemes for clinical trials needs to incorporate issues related to tumor heterogeneity in order to accurately stratify patients.


Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.Ochratoxin A (OTA), one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), UHPLC-Orbitrap-high resolution mass spectrometry (HRMS) and liquid chromatography-multiple stage mass spectrometry (LC-MS Aspergillus species notably A. melleus, A. auricomus, A. alliaceus, A. petrakii, as well as Penicillium verrucosum  m/z 358 a. m/z 578) of Compounds 2 and 3 were isobaric with the [M − H]− of Compound 1, and therefore exhibited the same ion formula C26H25O12NCl  product ions of the three compounds showed that besides the characteristic fragments (m/z 175 and 113) of glucuronate moieties, an intense peak at m/z 193 corresponding to the glucuronate anion was observed in the spectra of Compound 3  method was established using the obtained standards . The tra−1), with the parameters indicated in in vivo, was hardly found in incubations with liver microsomes, since hydrolysis is known to occur in the gut. When peptide cleavage occurs by an unspecific hydrolytic activity in the reaction mixture, OTα is then apparently further conjugated by microsomal UGTs in Reactions 1 and 2. As hydrolysis but not conjugation could occur in Reaction 3, however, a low content of OTα was also found in Mixture 3.As reported, OTα is a very important metabolite ,44. Howein vitro metabolic profile of OTA via biotransformation by rat liver microsomes is proposed in Based on the above observations, the N-acetylgalactosamine disodium salt (UDPAG), anhydrous magnesium chloride (MgCl2) and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were from Sigma-Aldrich . β-glucuronidase and microsomes  were from Sigma-Aldrich .Methanol and acetonitrile were ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) grade from Biosolve . Water was purified by a Milli-Q system . Ammonium acetate (AmAc), ammonium formate (AF), formic acid (FA), uridine 5’-diphosphoglucuronic acid trisodium salt (UDPGA), uridine 5’-diphospho-−1 of stock solutions.Ochratoxin A (OTA) and ochratoxin α (OTα) were from Coring System Diagnostix GmbH . Accurately weighed solid portions of OTA and OTα standards were dissolved in acetonitrile to prepare 0.5 mg mLThe OTA methyl ester was prepared according to the previous report with a minor modification . The OTA® H-class . The compounds were separated on an Acquity® UPLC HSS T3 column  at 40 °C, with a mobile phase flow rate of 0.5 mL min−1. The mobile phase consisted of (A) 10 mmol L−1 ammonium acetate solution, (B) water and (C) methanol. A linear gradient elution program was applied as follows: 0 min 1% A and 29% C, 10 min 1% A and 59% C, 10.2 min 1% A and 99% C, 11 min 1% A and 99% C, 11.8 min 1% A and 29% C, and hold on for a further 2.2 min for re-equilibration, giving a total run time of 13 min. The injection volume was 5.0 μL . The LC system consisted of an Acquity UPLC® mass spectrometer (Waters) was used for the analysis of the target compounds. Full scan analysis was performed both in the positive electrospray ionization mode (ESI+) and the negative electrospray ionization (ESI−) mode. The following settings were used: source temperature, 150 °C; desolvation temperature, 500 °C, scan range, m/z 100–1000, inter-scan delay, 0.01 s. The cone and desolvation gas flows were 30 and 1,000 L h−1, respectively. Data acquisition and processing were performed using MassLynx v4.1 (Waters). A XEVO TQ-STM Benchtop Orbitrap mass spectrometer  analysis in the full scan mode  was utilized for the metabolic profiling study. Chromatographic separation was achieved on a Zorbax Eclipse Plus C18 column  at 30 °C, with a mobile phase flow rate of 0.4 mL min−1. The mobile phase consisted of (A) water/methanol  containing 0.1% formic acid and 10 mM ammonium formate and (B) water/methanol  containing 0.1% formic acid and 10 mM ammonium formate. A linear gradient elution program was applied as follows: 0 min 0% B, 0.5 min 0% B, 20 min 99.1% B, 21 min 99.1% B, 24 min 0% B and hold on for a further 4 min for re-equilibration, giving a total run time of 28 min. The injection volume was 5.0 μL. The mass spectrometer was operated both in HESI+ and in HESI−. The following settings were used: spray voltage, 4.5 kV; capillary temperature, 250 °C; heater temperature, 250 °C; sheath gas flow, 45 a.u.; auxiliary gas, 10 a.u; sweep gas, 2 a.u., resolution 100000 FWHM at 1 Hz (1 scan per second). The automatic gain control (AGC) target was set at high dynamic range (3e6), and the maximum injection time was 20 ms. Initial instrument calibration was achieved by infusing calibration mixtures (Thermo Fisher Scientific) for positive and negative ion modes. The positive calibration mixture included caffeine, Met-Arg-Phe-Ala acetate salt (MRFA) and Ultramark 1621®, while the negative calibration solution comprised sodium dodecyl sulfate, sodium taurocholate and Ultramark 1621®. These compounds were dissolved in a mixture of acetonitrile, water and methanol, and both mixtures were infused with a Chemyx Fusion 100 syringe pump (Thermo Fisher Scientific). Data were acquired and processed by Xcalibur 2.1 and Sieve 2.0 software .UHPLC-Exactive+ and in HESI− with the following settings: source voltage of 5 kV, capillary temperature of 250 °C; heater temperature of 175 °C; sheath gas flow of 45 a.u.; aux gas of 10 a.u. The Xcalibur 2.0.7 software (Thermo Scientific) was used for instrument control, data acquisition and processing.HPLC-ion-trap-MS system (Thermo Fisher Scientific) was used for the fragments analysis of the targeted analytes. The column used was an X-bridge C18 column , supplied by Waters . The mobile phase consisted of (A) water/methanol  containing 0.1% formic acid and 10 mM ammonium formate and (B) water/methanol  containing 0.1% formic acid and 10 mM ammonium formate. The linear gradient elution program for LC-Ion-trap analysis was: 0–1 min B = 50%, 1–13 min B = 50%–97%, 13–18 min B = 97%, 18–19 min B = 97%–50%, and hold on for a further 6 min for re-equilibration, giving a total run time of 25 min. The mass spectrometer was operated both in HESIin vitro metabolic study of OTA via glucuronidation biotransformation by rat liver microsomes  was performed based on a slightly modified procedure adopted from Wu et al. [et al. [The u et al.  and Wels [et al.  for syntReaction 1: An aliquot of OTA stock solution  was transferred into a 5 mL tube and dried by nitrogen gas under 40 °C. Next, 15 μM of UDPGA, 0.25 μM of UDPAG and 5 μM of MgCl2 were added and all these reagents were dissolved in 400 μL of 50 mM Tris-HCl buffer (pH = 7.4). Rat liver microsomes (100 μL) were firstly mixed with 5 μg of alamethicin and kept on ice for 10 min before adding into the tubes. Then, the 500 μL reaction mixture was inverted a few times and incubated at 37 °C in a water bath for 1.5 h. The reaction was terminated by addition of 1 mL of methanol, vortexed for 30 s and centrifuged at 14,000 rpm for 10 min. The supernatant (100 µL) was diluted with 900 μL of methanol/water solution  and was ready for injection.Reaction 2: An aliquot of OTA stock solution  was transferred to a 5 mL tube and dried by nitrogen gas under 40 °C. Next, 15 μM of UDPGA, 0.25 μM of UDPAG and 5 μM of MgCl2 were added and all these reagents were dissolved in 400 μL of 50 mM Tris-HCl buffer (pH = 7.4). Then 100 μL  of rat liver microsomes were added. The reaction mixture (500 μL) was subsequently pretreated as reaction 1.Reaction 3: A blank reaction was performed using the same procedure and ingredients as in reaction 2, except for UDPGA. Reaction 4: A control reaction was also performed using the same procedure and ingredients as in Reaction 2, except for OTA. E. coli, Sigma-Aldrich, Saint Louis, MO, USA) at 37 °C in 0.25 mL of 0.1 M NaAc buffer (pH = 5) for 18 h. The reaction was terminated by addition of 1 mL methanol, vortexed for 30 s and centrifuged at 14,000 rpm for 10 min. The supernatant was passed through a 0.22 μm nylon filter and was ready for injection Hydrolysis of the reaction solution was performed for further identification of the OTA-glucuronides. An aliquot of the reaction solution (10 μL) was incubated with β-glucuronidase (1.5 units/reaction) (type IX, from in vitro biotransformation pathway of OTA in rat microsomes was proposed. The results obtained here will help to have deeper understanding on the theoretical basis of OTA clinical toxicology.To address the remaining uncertainties regarding OTA biotransformation by liver microsomes, UHPLC-MS/MS, UHPLC-Orbitrap-HRMS and LC-ion-trap were applied as combined strategies to investigate the metabolic profile of OTA via glucuronidation by rat liver microsomes. Three different OTA glucuronide conjugates, which corresponded to OTA amino-glucuronide, OTA phenol-glucuronide and OTA acyl-glucuronide were clearly identified. The suggested structures were supported by the fragments observed in the mass spectrometers and by hydrolysis with β-glucuronidase. OTA methyl ester, OTα and OTα-glucuronide were formed in the same reaction mixture. A possible


Klebsiella oxytoca, a Gram-negative, rod-shaped, and facultative anaerobic bacterium, is one of the most promising 2,3-butanediol  producers. In order to improve the metabolic performance of K. oxytoca as an efficient biofactory, it is necessary to assess its metabolic characteristics with a system-wide scope, and to optimize the metabolic pathways at a systems level. Provision of the complete genome sequence of K. oxytoca enabled the construction of genome-scale metabolic model of K. oxytoca and its in silico analyses.K. oxytoca was constructed using the annotated genome with biochemical and physiological information. The stoichiometric model, KoxGSC1457, is composed of 1,457 reactions and 1,099 metabolites. The model was further refined by applying biomass composition equations and comparing in silico results with experimental data based on constraints-based flux analyses. Then, the model was applied to in silico analyses to understand the properties of K. oxytoca and also to improve its capabilities for 2,3-BD production according to genetic and environmental perturbations. Firstly, in silico analysis, which tested the effect of augmenting the metabolic flux pool of 2,3-BD precursors, elucidated that increasing the pyruvate pool is primarily important for 2,3-BD synthesis. Secondly, we performed in silico single gene knockout simulation for 2,3-BD overproduction, and investigated the changes of the in silico flux solution space of a ldhA gene knockout mutant in comparison with that of the wild-type strain. Finally, the KoxGSC1457 model was used to optimize the oxygen levels during fermentation for 2,3-BD production.The genome-scale metabolic model of K. oxytoca at systems level. The KoxGSC1457 model could be employed as an useful tool to analyze its metabolic capabilities, to predict its physiological responses according to environmental and genetic perturbations, and to design metabolic engineering strategies to improve its metabolic performance.The genome-scale metabolic model, KoxGSC1457, constructed in this study successfully investigated metabolic characteristics of  Klebsiella oxytoca is known as one of the most promising 2,3-BD producers [K. oxytoca KCTC1686 consists of a chromosome of 5,974,109 bp with a 56.05% GC content, including 5,488 coding genes [K. oxytoca E718 is composed of a chromosome of 6,097,032 bp and two plasmids of 324,906 bp and 110,781 bp with a 55.5% GC content, including 5,909 coding genes [K. oxytoca KCTC12133BP from a cattle farm and sequenced its whole genome, but the sequence information, which has not yet been published, consists of a chromosome of 5,903,932 bp and a plasmid of 109,773 bp with a 55.4% GC content, including 5,793 coding genes.There is growing interest in the production of 2,3-butanediol  by microbial fermentation, as it can be easily converted to methyl ethyl ketone and tetramethyl ether, blending agents for gasoline, and 1,3-butadiene, an intermediate in synthetic rubber manufacture . Klebsieroducers , and itsng genes . The genng genes . More reK. oxytoca is to produce large amounts of C3/C4 diols, 1,3-propanediol  and 2,3-BD, using various carbon sources [K. oxytoca coproduced 83.6 and 60.1 g/L of 1,3-PD and 2,3-BD, respectively, in a fed-batch fermentation utilizing mixed substrates of glycerol and sucrose [K. oxytoca also could produce more than 95 g/L of 2,3-BD from glucose with a yield of 0.478 g/g  and a productivity of 1.71 g/L/h in a batch fermentation, in which the agitation speed was switched from 300 to 200 rpm during the fermentation [K. oxytoca significantly affects its physiology and 2,3-BD production [K. oxytoca is to readily metabolize glycerol, an inevitable by-product of biodiesel production, into biomass and products of value [The most important characteristics of  sources ,13-20. Fentation . The avaoduction -23. Anotof value ,25.K. oxytoca an attractive host for industrial applications. Furthermore, the U.S. National Institute of Health  has reported that K. oxytoca belongs to risk group 1 (RG 1), recognizing it as a GRAS  organism. However, in order to use this organism on an industrial scale, the strain should be further developed. Systems metabolic engineering allows the rational design of metabolic networks for the overproduction of target compounds and the creation of industrially useful microorganisms [in silico genome-scale metabolic model of K. oxytoca, KoxGSC1457, was constructed based on genome information, databases, and experimental data. The KoxGSC1457 model is composed of 1,457 reactions and 1,099 metabolites  was completed, 1,457 metabolic reactions and 1,099 metabolites were included in the final construction  and CO2 in the presence of oxygen. Diacetyl can then be catalyzed into acetoin (C4H8O2), which is the precursor of 2,3-BD, by diacetyl reductase (E.C. 1.1.1.303). K. oxytoca has its specialty for glycerol utilization by oxidative and reductive routes. Thus, the model encompasses the reactions for glycerol utilization, which are catalyzed by glycerol kinase (E.C. 2.7.1.30) and glycerol-3-phosphate dehydrogenase (E.C. 1.1.5.3) of the oxidative route, and NAD-dependent glycerol dehydrogenase (E.C. 1.1.1.6) and dihydroxyacetone kinase (E.C. 2.7.1.29) of the reductive route. Finally, the refined KoxGSC1457 model was used to understand the metabolic characteristics of K oxytoca and was applied to analyze the strategies for 2,3-BD production.The KoxGSC1457 model contains the reactions for 2,3-BD biosynthesis catalyzed by acetolactate synthase (E.C. 2.2.1.6), acetolactate decarboxylase (E.C. 4.1.1.5), and acetoin reductase (E.C. 1.1.1.4). The model also includes a spontaneous reaction that converts α-acetolactate into diacetyl . 2-acetolactate is then transformed into acetoin by decarboxylation to CO2 (budA). 2,3-BD is then synthesized from acetoin with oxidation of NADH into NAD+ by acetoin reductase (budC) . As a result, the knockout of ldhA gene encoding lactate dehydrogenase, which converts pyruvate into lactic acid with NADH oxidation, was targeted with top priority for a single gene knockout strategy based on the criteria of the maximization of 2,3-BD production rate and minimization of byproducts formation rates. Then, the KoxGSC1457 model investigated the metabolic characteristics of the ldhA gene knockout mutant by examining the changes of flux solution space compared with those of the wild-type strain  level in medium is known as one of the most important environmental factors [K. oxytoca, the oxygen uptake rate was perturbed by constraints-based flux analysis between 0 and 30 mmol/gDCW (gram dry cell weight)/h and glucose uptake rate was fixed to 10 mmol/gDCW/h, with maximization and minimization of 2,3-BD production rate as the objective function  during the whole period of fermentation. The fermentations were performed by using the ldhA gene knockout mutant of K. oxytoca, as the most productive 2,3-BD producer up to this point. As a result, the final 2,3-BD concentration (g/L) and yield (g/g glucose) increased through increasing the agitation speed to 350 rpm, but further increasing agitation speed caused a decrease of the final 2,3-BD concentration and yield. Meanwhile, the maximal value of 2,3-BD productivity of 2.1 g/L/h was observed at 450 rpm. The highest glucose consumption was observed at 550 rpm, however, most consumption of carbon source was used to synthesize not 2,3-BD but biomass at 550 rpm and agitation speeds greater than 550 rpm. In addition, fermentation at 10% DO level showed less 2,3-BD concentration, yield, and productivity than at 5% DO. These results are a strong indication that excessive oxygen exposure during fermentation impairs 2,3-BD overproduction. Since the optimal oxygen level exists beyond the scope of DO as represented in Figure This simulation was validated by batch fermentation experiments under different agitation speeds (rpm) and DO levels Figure B and C. K. oxytoca is a promising microorganism which has great potential in the industrial production of 2,3-BD. To make the better use of this organism and to improve industrial applicability, we constructed a genome-scale metabolic model of K. oxytoca, KoxGSC1457, consisting of 1,457 reactions and 1,099 metabolites based on information obtained from genome annotation data, database, literature and validation experiments. Using the KoxGSC1457 model, in silico metabolic fluxes were analyzed to investigate the effects of environmental and genetic variation. First, the effects of increasing the metabolic flux pools of 2,3-BD precursors were examined to understand the most important precursor to be augmented for 2,3-BD overproduction. In silico single gene knockout simulation was then performed to identify gene knockout candidates for the reduction of byproduct formation, and the change of flux solution space was then analyzed by elimination of ldhA gene resulting in the K. oxytoca ldhA gene knockout mutant, which is one of organisms that shows best performance for 2,3-BD production so far. Finally, to design strategies for better 2,3-BD overproduction, in silico metabolic flux analyses of KoxGSC1457 were executed to examine the effects of varying oxygen uptake rate during 2,3-BD production, and to determine the optimal agitation speed. Through the successful use of the KoxGSC1457 genome-scale metabolic model like those, it gave us the confidence that the model was thoroughly validated in comparison with various experimental data. There are still many difficulties associated with the overall economics of producing 2,3-BD through K. oxytoca on an industrial scale. Hence, it is expected that the model constructed in this study will support us to understand cellular physiology on systems level, broaden our insight on this organism, and conquer problems more systematically.K. oxytoca were collected from various sources, such as experiments, literatures, and public databases, in order to reconstruct the genome-scale metabolic model of K. oxytoca. First, information of genome annotation was gained from genome data in NCBI (http://www.ncbi.nlm.nih.gov/). Then, the biochemical reactions of K. oxytoca were assembled from various literatures, experiments, and public databases, including the Kyoto Encyclopedia of Genes and Genomes , Biocycs . The neutral and amino sugars were extracted from cells by treating them with 2 M trifluoroacetic acid and 6 N HCl, respectively, at 100°C for 4 hours. The carbohydrates were analyzed by ICD-5000  equipped with a CarboPac PA10 column  and a CarboPac PA10 cartridge (4 × 50 mm). 16 mM NaOH was used as a mobile phase with a flow rate of 1.0 mL∙ min-1. Then, data were analyzed by Chromelon ver 6.8 software. Amino acid compositions were analyzed by a Hewlett Packard 1100 series HPLC systems equipped with Waters Nova-Pak C18 4 um column (3.9 × 300 mm) . The fatty acid composition of K. oxytoca was determined by Sherlock microbial identification system of Sherlock version 6.1 . DNA and RNA compositions were analyzed based on genome information of K. oxytoca. The DNA composition was calculated by using the GC content (53%) of K. oxytoca. The RNA composition was calculated based on nucleotide sequences for rRNA and tRNA derived from genome information. The compositions for other components were obtained from literature or assumed reasonably described in Additional file The composition of biomass components was experimentally measured to construct the biomass formation reactions of in silico simulations, and to predict the metabolic characteristics of K. oxytoca, constraints-based flux analysis, including FBA, was carried out under the assumption of a pseudo-steady state [in silico model more accurately, the limits of uptake and secretion rates for some metabolites, including amino acids and organic acids such as acetic acid, ethanol, formic acid, lactic acid, pyruvic acid, and succinic acid, were constrained by experimentally measured flux values. Likewise, the limits related with secretion for some metabolites, which were not produced during fermentation such amino acids, were constrained to zero.In order to perform dy state ,40-43. IThe effect of 2,3-BD production rate in response to varying oxygen uptake rate was examined by flux response analysis -46. The 600). The tube and flask cultivations were placed in a rotary shaker at 150 rpm and 37°C (JEIO Tech. Co. SI-900R). The culture medium contained per liter: yeast extract , 5 g; FeSO4 · 7H2O, 0.05 g; ZnSO4 · 7H2O, 0.001 g, MnSO4 · H2O, 0.001 g; CaCl2∙ 2H2O, 0.001 g; MgSO4∙ 7H2O, 0.25 g; (NH4)2SO4, 6.6 g; K2HPO4, 8.7 g; KH2PO4, 6.8 g; trace metal solution, 10 mL. The trace metal solution contains per liter: FeSO4∙ 7H2O, 5 g; ZnSO4∙ 7H2O, 0.1 g; MnSO4∙ H2O, 0.1 g; CaCl2∙ 2H2O, 0.1 g; HCl 10 mL.For seed preparation, suspended cells from single colonies on Luria-Bertani (LB) agar  plates were precultured in 20 mL test tubes containing 5 mL culture medium at 37°C for 5 h. 1 mL aliquots of the preculture were then transferred to 500 mL Erlenmeyer flasks containing 300 mL culture medium, and the cells were cultivated to an optical density of 1.5 ~ 2.0 at 600 nm (OD-1∙ minute-1). The temperature was maintained at 37°C. The pH was controlled at 6.5 ± 0.1 by the automatic feeding of 5 N NaOH. Foaming was controlled by the addition of Antifoam 289 . For biomass composition analysis, the cells were cultured in a medium in which only yeast extract was removed from the culture medium. All fermentations were performed at least three times independently, and the representative results are shown in Figures.Batch fermentations were performed in a 5-L BIOFLO & CELLIGEN 310 bioreactor  with 3 L culture medium containing 90 g/L of D-glucose and 300 mL seed culture. The fermenter was continuously aerated through a 0.2 μm membrane filter at a flow rate of 1 vvm  to monitor cell growth. Cell concentration, DCW per liter of culture broth, was calculated from the pre-determined standard curve relating OD600 to DCW (1 OD600 = 0.3877 ± 0.0136 g DCW∙ L-1). DCW was determined by filtering the culture broth through a 0.45 μm membrane and washing it twice with an equal volume of deionized distilled water. The filtered cells were then dried at 80 ± 5°C overnight and cooled to room temperature in a desiccator prior to weighting.The concentrations of D-glucose and metabolites, including 2,3-BD, formic acid, ethanol, acetic acid, lactic acid, succinic acid, and acetoin, were determined by a high-performance liquid chromatography equipped with UV/VIS and RI detectors . An Aminiex HPX-87H column  was isocratically eluted with 0.01 N H-1• minute-1; gDCW: Gram dry cell weight; DO: Dissolved oxygen; rpm: Revolutions per minute1,3-PD: 1,3-propanediol; 2,3-BD: 2,3-butanediol; ORF: Open reading frame; FBA: Flux balance analysis; KEGG: Kyoto encyclopedia of genes and genomes; LB: Luria-bertani; vvm: Air volume• working volumeThe authors declare that they have no competing interests.JMP, HS, HJL, and DS generated ideas. JMP and HS designed the research. JMP performed the research. JMP performed analytical experiments. JMP analyzed data. JMP, HS, HJL, and DS wrote the paper. All authors read and approved the final manuscript.List of metabolic reactions in the genome-scale metabolic model of Klebsiella oxytoca.Click here for fileList of metabolites in the genome-scale metabolic model of Klebsiella oxytoca.Click here for fileBiomass composition of Klebsiella oxytoca.Click here for file


Positive developmental impact of low stress-induced glucocorticoid levels in early development has been recognized for a long time, while possible involvement of mineralocorticoids in the stress response during the perinatal period has been neglected. The present study aimed at verifying the hypothesis that balance between stress-induced glucocorticoid and mineralocorticoid levels is changing during postnatal development. Hormone responses to two different stressors  measured in 10-day-old rats were compared to those in adults. In pups corticosterone responses to both stressors were significantly lower than in adults, which corresponded well with the stress hyporesponsive period. Importantly, stress-induced elevations in aldosterone concentration were significantly higher in pups compared both to corticosterone elevations and to those in adulthood with comparable adrenocorticotropin concentrations in the two age groups. Greater importance of mineralocorticoids compared to glucocorticoids in postnatal period is further supported by changes in gene expression and protein levels of gluco- (GR) and mineralocorticoid receptors (MR) and selected enzymes measured by quantitative PCR and immunohystochemistry in the hypothalamus, hippocampus, prefrontal cortex, liver and kidney. Gene expression of 11beta-hydroxysteroid dehydrogenase 2 (11β-HSD2), an enzyme enabling preferential effects of aldosterone on mineralocorticoid receptors, was higher in 10-day-old pups compared to adult animals. On the contrary, the expression and protein levels of GR, MR and 11β-HSD1 were decreased. Presented results clearly show higher stress-induced release of aldosterone in pups compared to adults and strongly suggest greater importance of mineralocorticoids compared to glucocorticoids in stress during the postnatal period. Aldosterone is the main mineralocorticoid hormone involved in the control of water-electrolyte balance In stress research, less attention has been given to aldosterone compared to cortisol and corticosterone  mainly because they are acting on the same receptors and glucocorticoids are the dominant players. They have two types of receptors, namely the low affinity glucocorticoid receptor (GR) and the high affinity mineralocorticoid receptor (MR) Since concentration of circulating glucocorticoids is 2–3 order higher than that of aldosterone, in aldosterone target tissues such as in the kidney, only the presence of 11-beta-hydroxysteroid dehydrogenase type 2 (11β-HSD2) allows steroid binding to receptors via conversion the competing corticosterone and cortisol into inactive metabolites Effects of stress-induced aldosterone release on the salt-water homeostasis have impact on the control of blood pressure and cardiovascular functions as well Another important issue could be the involvement of aldosterone in neuroendocrine responses to stress stimuli during the postnatal period. Even though there are reports indicating that postnatal stress may have implications on the salt intake in the adulthood In the present studies, we aimed to test the hypothesis that the balance between stress-induced glucocorticoid and mineralocorticoid levels is changing during development. More specifically, we hypothesized that during SHRP, which is associated with decreased glucocorticoid secretion, mineralocorticoids are the dominant adrenocortical steroids released during stress.Adult  and postnatal  male Wistar rats were investigated . Rats were kept in controlled environment  and given commercial rat chow  and tap water ad libitum.The experiments were performed in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC), and were reviewed and approved by the Animal Welfare Committee of the Institute of Experimental Medicine (MÁB 22.1/2654/003/2007).Stress of hypoglycaemia was induced in fasted rats by intraperitoneal (i.p.) insulin injection  at the dose of 3IU/1 ml/kg of body weight in saline. Adult rats were fasted for 18 h, while 10-day-old pups for only 4 h  to achieve comparable degree of starvation. Rats of control groups were injected with the same volume of vehicle (0.9% NaCl). Separate groups of rats were decapitated at 60 and 90 min after i.p. injection. As the HPA axis of pups reacts to different stimuli with a delay, we took the 90 min results as more representative and presented these results in the graphic form. Data obtained at 60 min are in a table. Blood glucose levels were measured by commercially available analyser  at the time of decapitation.Administration of LPS represents a strong stressor simulating bacterial infection. Escherichia coli LPS, serotype 0111: B4  was used at the dose of 100 μg/1 ml/kg of body weight i.p. Rats of control groups were injected with the same volume of vehicle (0.9% NaCl). Animals were killed by decapitation 120 min after the injection.2–5% CO2 atmosphere for 60 min. Thereafter the buffer was replaced with fresh DMEM, and the adrenals were preincubated for additional 60 min. Following the preincubation, 15-min samples were collected three times adding 10−10 M ACTH into the second fraction. At the conclusion of the experiment, media were removed, centrifuged at 3000 g for 5 min and the supernatant was stored at −20°C until corticosterone and aldosterone measurements. The total secreted amount during the whole observation period (3×15 min) was expressed as area under the curve (AUC).Incubation followed the method of Stachura et al. Trunk blood was collected by decapitation into ice-cold plastic tubes, centrifugated at 2000 g for 20 min at 4°C and the serum was stored at −20°C until analysed. Because of the small amount of blood in postnatal rats, separate pups were used for ACTH/corticosterone and for plasma renin activity/aldosterone measurements. Samples from a particular experiment were always assessed in the same RIA.Plasma ACTH concentrations were measured by radioimmunoassay (RIA) in 50 μl of unextracted plasma as described earlier CT experiments were designed by the Primer Express 3.0 program. Melting curve analysis to confirm the identity of PCR products has been performed by using ABI StepOnePlus instrument's Software v.2.1 according to the instructions of the manufacturer. Gene expression was analyzed by ABI StepOne Software 2.1 program. The primers were the following:Tissue samples of macrodissected brain regions, kidney and liver were collected under RNase free conditions from unstressed adult and postnatal rats and were kept at −70°C. Total RNA was isolated from homogenates of the hypothalamus, pituitary, hippocampus, prefrontal cortex, kidney and liver using RNeasy Mini Kit  and then converted to cDNA by High-Capacity cDNA Reverse Transcription Kit . The cDNA samples were pooled for each group and measured in duplicates. Real-time PCR was performed using Power SYBR Green PCR Master Mix (Life Technologies) on ABI StepOnePlus instrument according to the manufacturer's instructions. Primers used for the comparative 5′-TCC TGG GTT GTG TCA TGA ACA-3′; GAPDH forward: 5′-ACA GCC GCA TCT TCT TGT GC-3′, reverse: 5′-GCC TCA CCC CAT TTG ATG TT-3′.GR forward: 5′-CAT CTT CAG AAC AGC AAA ATC GA-3′, reverse: 5′-AGG TGC TTT GGT CTG TGG GAT A-3′; MR forward: 5′-CCA AGG TAC TTC CAG GAT TTA AAA AC-3′, reverse: 5′-AAC GAT GAT AGA CAC ATC CAA GAA TAC T-3′; 11β-HSD1 forward: 5′-CCT CCA TGG CTG GGA AAA T-3′, reverse: 5′-AAA GAA CCC ATC CAG AGC AAA C-3′; 11β-HSD2 forward: 5′-CGC CGC TTC CTA CAG AAC TT-3′, reverse: −ΔΔCT method.GAPDH was used as endogenous control. Relative quantity of mRNAs was referred to corresponding samples of the adult Wistar rats based upon the 22O2 solution and again 3×5 min in PBS. Then sections were incubated in 2% Normal Goat Serum  for 60 min. The GR and MR proteins were immuno labeled with rabbit polyclonal antibodies raised against GR and MR  for 48 h at 4°C. This was followed by incubation in biotinylated anti-rabbit serum . The antigens were then visualized by conventional Avidin-Biotin-HRP technology  and developed using 0.05% diaminobenzidine  and 0.01% H2O2 in PBS. Sections were then mounted onto geletine coated slides, dehydrated and coverslipped. Images were taken with a digital camera  coupled to a bright-field microscope , using a 20× objective, with no further modifications. The following brain areas were investigated: hypothalamus , prefrontal cortex, hippocampus. Representative photomicrographs are shown on figures.Animals were anesthetized at rest by pentobarbital  and perfused transcardially with saline solution (0.9% NaCl) for 2 min, then with 30 mL (pups) or 300 mL (adult) ice cold fixative . The brains were removed, post-fixed in the fixative for 3 h then placed in PBS 10% sucrose overnight at 4°C. On the next day 30 μm sections were cut in the coronal plane on a freezing microtome and were stored at −20°C in cryoprotectant. The sections were washed first in PBS for 3×10 min and for blocking the endogen peroxidase in HValues are presented as mean ± SEM. Data were analysed by analysis of variance using one (age) or two way ANOVA  of the STATISTICA 11.0 software package . Multiple pairwise comparisons where appropriate were made by the Newman–Keuls method. Results of the post hoc analysis are presented on the figures. In case there was no significant interaction, the main effects are indicated in the text only. = 223.00, p<0.01)  = 524.0, p<0.01)  = 12.1, p<0.01) there was no significant difference in the degree of hypoglycaemia observed in postnatal and in adult rats (no significant interaction between age x treatment). Concentrations of ACTH increased in the stressed groups  = 4.53, p<0.01)  = 58.04, p<0.01)  = 311.96, p<0.01; treatment F = 305.84, p<0.01; age x treatment F = 254.96, p<0.01)  = 202.63, p<0.01; treatment F = 233.80, p<0.01 age × treatment F = 130.70, p<0.01)  = 5.59, p<0.05)  = 18.96, p<0.01)  = 16.95, p<0.01)  = 10.46, p<0.01; age × treatment F = 4.51, p<0.05) (Blood glucose levels decreased significantly at 60 minutes (treatment: F p<0.01) , and 90  p<0.01)  followin p<0.01) ; (at 90  p<0.01)  in a sim p<0.01)  and 90 m p<0.01)  followin p<0.05) . On the  p<0.01) , but als p<0.01) . At 90 m p<0.05) . = 31.90, p<0.01)  = 784.84, p<0.01; treatment F = 1120.68, p<0.01; age × treatment F = 592.04, p<0.01)  = 41.74, p<0.01), exhibiting the highest values in LPS treated pups, which were significantly different from those in LPS injected adults  = 36.10, p<0.01; age × treatment F = 26.12, p<0.01)  = 30.96, p<0.01; treatment F = 60.64, p<0.01; age × treatment F = 24.55, p<0.01)  . Concent p<0.01) . Plasma  p<0.01) . Changes p<0.01) . = 155.27, p<0.01; hippocampus: F = 329.69, p<0.01; prefrontal cortex: F = 39.80, p<0.01; liver: F = 95.89, p<0.01; kidney: F = 76.21, p<0.01)  = 385.75, p<0.01; hippocampus: F = 74.04, p<0.01; prefrontal cortex: F = 412.66, p<0.01; liver: F = 215.62, p<0.01; kidney: F = 227.99, p<0.01)  . Similar p<0.01) . = 168.39, p<0.01; prefrontal cortex: F = 56.65, p<0.01), while there was no age effect in the hypothalamus  = 11092.12, p<0.01)  =  50.98, p<0.01; hippocampus: F =  249.61, p<0.01; kidney: F =  1815.96, p<0.01)  . In cont p<0.01) . No age GR immunoreactive (ir) neurons were widely distributed in the brains of both adults and pups. Their distribution in the hypothalamus  and in prefrontal cortex region is presented in The MR proteins were found to be highly expressed in the hippocampal  and medi =  26,8, p<0.01)  = 5.2, p<0.05). However, the ACTH-induced corticosterone secretion was not different in the two age-groups (no interaction between age × treatment).The overall corticosterone secretion (AUC) was significantly lower in pups compared to adults during the whole observation period (age: F p<0.01) . Adminis = 34.8, p<0.01)  = 27.4, p<0.01), which was detectable only in adults  = 22.45, p<0.01).Regarding aldosterone secretion, a similar general diminution was visible in pups (age: F p<0.01) . ACTH adPresent data provide the first evidence of increased aldosterone responses to stress stimuli during the postnatal period suggesting a shift in the balance between stress-induced glucocorticoid and mineralocorticoid hormone release during the development . In 10-dDampened stress-induced corticosterone concentrations in 10-day-old pups and facilitated aldosterone release were revealed using two different stress models , and different timing. The relatively low corticosterone release in response to stress stimuli is well described and it represents the essential characteristics of stress-hyporesponsiveness during the postnatal period The secretion of ACTH, which is an important secretagogue of aldosterone during stress, was similar in pups and adults in both stress models. During immune challenge, the other physiological regulator, angiotensin II Potential functional significance of enhanced aldosterone activity during postnatal period is supported by concomitant higher 11β-HSD2 and/or lower 11β-HSD1 mRNA levels in the kidney and several brain areas of pups compared to adults. The present study clearly shows increased mRNA expression of 11β-HSD2 and decreased expression of 11β-HSD1, GR and MR in the brain and kidney of 10-day-old pups compared to that in adult rats. In consistence with these findings, immunohistochemical analysis revealed similar differences in GR and MR protein levels. Developmental changes in gene expression and activity of 11β-HSD2 in some brain areas and peripheral tissues were reported during both fetal and postnatal periods It is suggested that the main physiological role of enhanced aldosterone secretion in pups is related to the maintenance of water-electrolyte balance Furthermore, MRs are present also in the heart, blood vessels, adipose tissue and macrophages Taken together, a primarily mineralocorticoid regulation under stress conditions at the time of postnatal SHRP may be evolutionarily warranted. Though additional information is needed, the results of the present study clearly show a higher stress-induced release of aldosterone in pups compared to adults and strongly suggest greater importance of mineralocorticoid compared to glucocorticoid actions during the postnatal period.


The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) , Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software . The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 ( Localiz2.2.1.The relative expression of AFP mRNA was significantly lower in albumin-added medium than in albumin-free medium and Prionex-added medium in both HepG2 and Hep3B cell lines in a concentration-dependent manner .The relative expression of p21 mRNA was significantly higher in albumin-added medium than in albumin-free medium and Prionex-added medium in HepG2 and Hep3B cell lines in a concentration-dependent manner , and theThe relative expression of p53 showed the same level in albumin-free medium, albumin-added medium, and Prionex-added medium in HepG2 and there was no p53 expression in Hep3B .2.2.Using media with 5 g/dL albumin, the effects of the albumin concentration on AFP, P21, P57 protein expressions were revealed. The relative expression of AFP protein expression was significantly lower in albumin-added medium than in albumin-free medium and Prionex-added medium in both HepG2 and Hep3B cell lines. We used cells cultured in serum-containing medium as a positive control and Kato III gastric cells that did not express AFP were used as a negative control. The same results were obtained for the protein level in p21 and p57 compared to mRNA expression.2.3.Protein expression levels of Rb were similar in all culture media. However, the phosphorylation of serine 807/811 and serine 780 of Rb protein was reduced in albumin-added medium and phosphorylation was recovered in Prionex-added medium.2.4.The effects of albumin concentration on the cell cycle were investigated using flow cytometry.Cell % populations in G0/G1, G2/M, and S-phase were 45.2 ± 1.1, 43.7 ± 0.5, and 11.2 ± 1.3 in albumin-free medium, 51.2 ± 1.1, 43.1 ± 0.6, and 5.8 ± 1.8 in albumin-added medium, and 44.8 ± 0.3, 43.1 ± 0.8, and 12.1 ± 1.1 in Prionex-added medium, respectively, using HepG2.Cell % populations in G0/G1, G2/M, and S-phase were 60.9 ± 0.6, 16.5 ± 0.9, and 22.6 ± 0.3 in albumin-free medium, 67.7 ± 1.3, 13.1 ± 0.2, and 19.2 ± 1.2 in albumin-added medium, and 61.5 ± 0.4, 15.6 ± 0.9, and 23.0 ± 0.5 in Prionex-added medium, respectively, using Hep3B.p <0.05).The G0/G1 phase in albumin-added medium had a significantly higher cell population percentage than in albumin-free and Prionex-added media in both HepG2 and Hep3B cell lines  containing 10% fetal bovine serum (FBS) and antibiotics. Twenty-four hours after seeding, the medium was changed to medium with no serum, containing 2 g/dL (some experiments), 5 g/dL human albumin  and 10% FBS for general control samples. Prionex  was added to generate the same osmotic pressure as albumin and its data were compared. Kato III gastric cancer cells were grown in RPMI-1640 medium containing 10% FBS to use as the negative control of AFP.4.2.O-vanadate, 5 mmol/L Na-pyrophosphate, and protease inhibitor cocktail . In some experiments, the same experiments were performed using 36 and 48 h incubation and compared to 24 h. After 10 min on ice, lysates were centrifuged in the cold for 5 min and either used immediately or stored at −70 °C until use. Tyrosine phosphorylated proteins were immunoprecipitated from protein-matched lysate samples during 4-h incubation with 15 μL antiphosphotyrosine-agarose conjugate . Immunoprecipitates were washed extensively with HNTG buffer , dissolved in Laemmli buffer, and placed in a boiling water bath for 5 min. Equal volumes of each sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5% gels and transferred to PVDF membranes. After blocking with 1% BSA in TBST  for 1 h at room temperature, antiphosphotyrosine immunoprecipitates were probed with anti-p780  and p807 antibodies , followed by anti-mouse IgG-horse radish peroxidase (HRPO) . Total cell lysates were directly diluted in Laemmli buffer, boiled, analyzed by gel electrophoresis, and probed with anti-AFP , P21  antibody and anti-Rb proteins (BD Biosciences) followed by anti-mouse IgG-horseradish peroxidase, and P57  followed by anti-rabbit antibody.After 24-h incubation with several media, cells were disrupted in lysis buffer containing 50 mM Tris (pH 8.0), 1 mM EDTA, 120 mM NaCl, 0.5% Triton X-100, 10% glycerol, 50 mmol/L NaF, 100 μmol/L Na-After extensive washing with TBST, membranes were treated with an enhanced chemiluminescence reagent .Bands were quantified densitometrically. Where required, the density of the phosphorylated bands and total Rb bands was normalized by comparison to β-actin  protein bands measured in the same experiment.4.3.Total RNA was extracted using Trizol reagent according to the manufacturer’s recommended protocol . RNA extracts were reverse-transcribed with random hexamers and avian myeloblastosis virus reverse transcriptase using a commercial kit . Expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated by real-time PCR using an ABI prism 7000 Sequence Detection system  according to the manufacturer’s protocol. Probes and primers for AFP (ID: Hs00609411_m1), p53 (ID: Hs99999147_m1), p21 (ID: Hs99399142_m1), and p57 (ID: Hs00175938_m1) were purchased from Applied Biosystems. The relative target was glyceraldehyde-2-phosphate dehydrogenase  mRNA in an identical cDNA sample using the standard curve method recommended by the manufacturer.4.4.To evaluate cell proliferation we counted cells after 24 h incubation in each solution using a Scepter handheld automated cell counter . Cells were cultured in several media in a 6 cm dish and collected by cleaving with trypsin to separate cells, washing twice with PBS, resuspending cells and then counting the cells.To estimate the cell toxicity of albumin and Prionex we measured lactate dehydrogenase (LDH) in the medium after 24-h culture using the Cyto Tox96 non-radioactive cytotoxicity assay , performed according to the manual.4.5.g cells were washed with PBS, resuspended in 0.9 mL PBS, then 2.1 mL of 100% EtOH was added, and cells were vortexed. Cells were incubated for 40 min on ice. After washing cells twice with PBS, 1 mL RNse A solution was added, and cells were incubated for 30 min at 37 °C. Cells were then centrifuged for 5 min at 1000× g, 1 mL PI solution was added, and cells were incubated for 20 min at room temperature (RT).For flow cytometric analysis of the cell cycle, FACS analysis was performed as described below: after 24 h incubation, cells were washed with PBS and incubated with 0.25% trypsin for 5 min at 37 °C. After centrifuging for 5 min at 1000× LT; BD, Topsham, ME, USA).The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACSCalibur (BD) with appropriate software (ModFit All experiments were performed using more than three individual trials and the significance of any intergroup differences was determined by ANOVA.5.The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.