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Invasive pulmonary aspergillosis (IPA) is a severe life-threatening condition. Diagnosis of fungal disease in general, and especially that caused by Aspergillus fumigatus is problematic. A. fumigatus secretes siderophores to acquire iron during infection, which are also essential for virulence. We describe the chemoacetylation of ferrated fusarinine C to diacetylated fusarinine C (DAFC), followed by protein conjugation, which facilitated triacetylfusarinine C (TAFC)-specific monoclonal antibody production with specific recognition of the ferrated form of TAFC. A single monoclonal antibody sequence was ultimately elucidated by a combinatorial strategy involving protein LC-MS/MS, cDNA sequencing and RNAseq. The resultant murine IgG2 a monoclonal antibody was secreted in, and purified from, mammalian cell culture (5 mg) and demonstrated to be highly specific for TAFC detection by competitive ELISA (detection limit: 15 nM) and in a lateral flow test system (detection limit: 3 ng), using gold nanoparticle conjugated- DAFC-bovine serum albumin for competition. Overall, this work reveals for the first time a recombinant TAFC-specific monoclonal antibody with diagnostic potential for IPA diagnosis in traditional and emerging patient groups (e.g., COVID-19) and presents a useful strategy for murine Ig sequence determination, and expression in HEK293 cells, to overcome unexpected limitations associated with aberrant or deficient murine monoclonal antibody production., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Currently there is a wide variety of collectors used in mineral processing, the xanthates being the most used in sulfides flotation. Unfortunately, it is known that xanthates are not stable compounds and their decomposition generates carbon disulfide (CS 2 ), a substance that is considered toxic. These aspects have motivated the search for collectors that exhibit superior performance without the health, safety and environmental (HSE) concerns associated with xanthates. In this study, the chemical stability of three xanthates of different alkyl groups (sodium ethyl xanthate (SEX), sodium isopropyl xanthate (SIPX) and potassium amyl xanthate (PAX)) was evaluated by UV/Vis spectroscopy, as a function of pH and time. Similarly, the chemical stability of three chelating collectors was evaluated: sodium di-isobutyl dithiophosphinate (SDIBDTPI), benzohydroxamic acid (BHA) and octanohydroxamic acid (OHA). Likewise, the surface tension of their aqueous solutions was measured making use of the Du Noüy method, to determine the critical micelle concentration (CMC). The results showed that the xanthate UV/Vis absorption spectra reflect the presence of a chemical reaction as the pH decreases from 4 to 2.5, which results in the formation of carbon disulfide (CS 2 ). In addition, the generation of CS 2 is favored as time elapses and the pH of the solutions decreases from 10 to 6, regardless of the hydrocarbon chain length. Conversely, dithiophosphinate and hydroxamic acids present greater chemical stability, although they form micelles at a certain concentration (CMC), a phenomenon that is not observed with xanthates. By not hydrolyzing, oxidizing, or decomposing into other chemical species, SDIBDTPI, BHA, and OHA may be considered environmentally friendly reagents. In the above context, it is important to promote the adoption of these collectors in mineral processing., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Studies on metabolomics and CKD have primarily addressed CKD incidence defined as a decline on eGFR or appearance of albuminuria in the general population, with very few evaluating hard outcomes. In the present study, we investigated the association between metabolites and mortality and ESRD in a CKD cohort., Setting and Methods: Data on 454 participants of the Progredir Cohort Study, Sao Paulo, Brazil were used. Metabolomics was performed by GC-MS (Agilent MassHunter) and metabolites were identified using Agilent Fiehn GC/MS and NIST libraries. After excluding metabolites present in <50% of participants, 293 metabolites were analyzed. An FDR q value <0.05 criteria was applied in Cox models on the composite outcome (mortality or incident renal replacement therapy) adjusted for batch effect, resulting in 34 metabolites associated with the outcome. Multivariable-adjusted Cox models were then built for the composite outcome, death, and ESRD incident events. Competing risk analysis was also performed for ESRD., Results: Mean age was 68±12y, mean eGFR-CKDEPI was 38.4±14.6 ml/min/1.73m2 and 57% were diabetic. After adjustments (GC-MS batch, sex, age, DM and eGFR), 18 metabolites remained significantly associated with the composite outcome. Nine metabolites were independently associated with death: D-malic acid (HR 1.84, 95%CI 1.32-2.56, p = 0.0003), acetohydroxamic acid (HR 1.90, 95%CI 1.30-2.78, p = 0.0008), butanoic acid (HR 1.59, 95%CI 1.17-2.15, p = 0.003), and docosahexaenoic acid (HR 0.58, 95%CI 0.39-0.88, p = 0.009), among the top associations. Lactose (SHR 1.49, 95%CI 1.04-2.12, p = 0.03), 2-O-glycerol-α-D-galactopyranoside (SHR 1.76, 95%CI 1.06-2.92, p = 0.03), and tyrosine (SHR 0.52, 95%CI 0.31-0.88, p = 0.02) were associated to ESRD risk, while D-threitol, mannitol and myo-inositol presented strong borderline associations., Conclusion: Our results identify specific metabolites related to hard outcomes in a CKD population. These findings point to the need of further exploration of these metabolites as biomarkers in CKD and the understanding of the underlying biological mechanisms related to the observed associations., Competing Interests: We have the following interests: Dr. Lotufo received horonaria from Abbot-Brazil, AbbVie-Brazil and Amgen for lectures. Dr. Thadhani is a consultant to Fresenius Medical Care North America. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

Hierarchical leaf-like gold nanolayers were electrodeposited using choline chloride as a shape directing agent and characterized using field emission scanning electron microscopy. The electrooxidation behavior of vorinostat was then studied on the nanolayers and the kinetic parameters of the electrodic process were obtained by voltammetric measurements in a phosphate buffer solution at pH 7.40. Vorinostat was electrooxidized on the nanolayers' surface at a lower potential and with a higher rate, compared to a polycrystalline smooth gold surface, through an irreversible process. Based on the results, an amperometric sensor was designed using the hierarchical leaf-like gold nanolayers for the determination of vorinostat. A linear dynamic range of 4.0-52μmol L -1 with a calibration sensitivity of 7.7mAmol -1 L, and a detection limit of 1.40μmolL -1 were obtained. The amperometry method was also applied to the analysis of vorinostat capsules., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Macrocyclic hydroxamic acids coordinate Fe(III) with high affinity as part of siderophore-mediated bacterial iron acquisition. Trimeric hydroxamic acid macrocycles, such as desferrioxamine E (DFOE), are prevalent in nature, with fewer dimeric macrocycles identified, including putrebactin (pbH 2 ), avaroferrin (avH 2 ), bisucaberin (bsH 2 ) and alcaligin (alH 2 ). This work used metal-templated synthesis (MTS) to pre-assemble complexes between one equivalent of Fe(III) and two equivalents of 4-((4-aminobutyl)(hydroxy)amino)-4-oxobutanoic acid (BBH) or 4-((5-aminopentyl)(hydroxy)amino)-4-oxobutanoic acid (PBH). Following peptide coupling, the respective Fe(III) complexes of pbH 2 or bsH 2 were formed, which analysed by LC-MS under acidic pH as [Fe(pb)] + ([M] + , m/z obs 426.1) or [Fe(bs)] + ([M] + , m/z obs 454.2). The mixed-ligand 1:1:1 Fe(III):BBH:PBH system furnished [Fe(pb)] + and [Fe(bs)] + , together with chimeric [Fe(av)] + ([M] + , m/z obs 440.2). The deviation from the expected 1:2:1 distribution of [Fe(pb)] + :[Fe(av)] + :[Fe(bs)] + to 1:3.2:1.6 suggested the MTS-mediated formation of dimeric macrocycles could be influenced by steric effects in the pre-complex and/or cavity size, as governed by the monomer. 21-Membered avH 2 defined the lower boundary of the optimal architecture. Mixed-ligand MTS between Fe(III):PBH-d 4 :ret-PBH at 1:1.5:1.5, where ret-PBH=3-(6-amino-N-hydroxyhexanamido)propanoic acid, gave four Fe(III)-loaded trimeric hydroxamic acid macrocycles in a distribution of 1.0:3.0:2.9:1.1 that closely matched the expected distribution 1:3:3:1 for a system without any kinetic and/or thermodynamic bias. Apo-macrocycles pbH 2 , avH 2 and bsH 2 were produced upon incubation with diethylenetriaminepentaacetic acid (DTPA) and co-eluted with a biosynthetic mixture of the native macrocycles. The work has demonstrated the utility of single- and mixed-ligand MTS for producing a variety of homo- and heteroleptic dimeric hydroxamic acid macrocycles as Fe(III) complexes and free ligands., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Effective iron acquisition and fine-tuned intracellular iron storage systems are the main prerequisites for a successful host invasion by a pathogen. Bacteria have developed several different strategies to sequester this essential element from their environment, one relies on the secretion of low molecular weight compounds with high affinity for ferric iron, the so-called siderophores. Here, we report hydroxamate siderophore structures produced by entomopathogenic bacteria of the species Xenorhabdus and Photorhabdus, which are known for their potential to produce bioactive natural products, required for their role as nematode symbiont and insect pathogen. Four siderophores could be identified, namely aerobactin, putrebactin, avaroferrin and ochrobactin C, which was found previously only in marine bacteria. While the putrebactin and avaroferrin producing biosynthesis gene cluster (BGC) is more widespread and most likely was present in a common ancestor of these bacteria, the aerobactin and ochrobactin producing BGC was probably taken up by a few strains individually. For aerobactin a role in virulence towards Galleria mellonella larvae is shown., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The use of gene expression signatures to classify compounds, identify efficacy or toxicity, and differentiate close analogs relies on the sensitivity of the method to identify modulated genes. We used a novel ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq®, to determine whether previously unreported compound-responsive genes could be identified and incorporated into a broad but specific compound signature. TempO-Seq exhibits 99.6% specificity, single cell sensitivity, and excellent correlation with fold differences measured by RNA-Seq (R2 = 0.9) for 20,629 targets. Unlike many expression assays, TempO-Seq does not require RNA purification, cDNA synthesis, or capture of targeted RNA, and lacks a 3' end bias. To investigate the sensitivity of the TempO-Seq assay to identify significantly modulated compound-responsive genes, we derived whole transcriptome profiles from MCF-7 cells treated with the histone deacetylase inhibitor Trichostatin A (TSA) and identified more than 9,000 differentially expressed genes. The TSA profile for MCF-7 cells overlapped those for HL-60 and PC-3 cells in the Connectivity Map (cMAP) database, suggesting a common TSA-specific expression profile independent of baseline gene expression. A 43-gene cell-independent TSA signature was extracted from cMAP and confirmed in TempO-Seq MCF-7 data. Additional genes that were not previously reported to be TSA responsive in the cMAP database were also identified. TSA treatment of 5 cell types revealed 1,136 differentially expressed genes in common, including 785 genes not previously reported to be TSA responsive. We conclude that TSA induces a specific expression signature that is consistent across widely different cell types, that this signature contains genes not previously associated with TSA responses, and that TempO-Seq provides the sensitive differential expression detection needed to define such compound-specific, cell-independent, changes in expression.

Siderophores are produced by a number of microbes to capture iron with outstandingly high affinity, which property also generates biomedical and industrial interests. Desferrioxamine E (DFO-E) secreted by streptomycetes bacteria can be an ideal candidate for iron chelation therapy, which necessitates its cost-effective production for in vitro and animal studies. This study focused on the optimization of DFO-E production by Streptomyces parvulus CBS548.68. Different combinations of various carbon and nitrogen sources as well as the addition of 3-morpholinopropane-1-sulfonic acid (MOPS) markedly affected DFO-E yields, which were attributed, at least in part, to the higher biomass productions found in MOPS-supplemented cultures. In MOPS-supplemented glucose and sodium glutamate medium, DFO-E productions as high as 2,009 ± 90 mg/l of culture medium were reached. High-performance liquid chromatography analysis demonstrated that a simple two-step purification process yielded DFO-E preparations with purities of ∼97%. Matrix assisted laser desorption ionization-time of flight mass spectrometry analysis showed that purified DFO-E always contained traces of desferrioxamine D2.

Oregano and thyme possess beneficial properties for human health, mainly attributable to monoterpenes such as thymol and carvacrol. The main objective of this research was to assess, on starchy food, the impact of cooking (boiling and baking) and delivery (ground leaves and essential oil) modes on retention and bioaccessibility of thymol and carvacrol. Retention was assessed after cooking, while bioaccessibility was estimated in cooked samples using an in vitro digestion model. Our results indicate that bioaccessibility was weakly dependent on cooking and delivery modes (27-33%). Boil cooking presented 20% more retention than baking for both compounds. When essential oil was added to the food matrix, thymol was retained almost 25% more when compared with ground leaves' addition. Conversely, carvacrol was retained 39% more when ground leaves were added., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro-fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10 μM) had the highest level of blastocyst development at 43.9%, and all treatments except 10 μM ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0 μM ISAHA and 1.0 μM SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10 μM SAHA- and 1 μM ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT.

The expression of aerobactin accounts for much of the pathogenesis of avian pathogenic Escherichia coli (APEC). iucA, iucB, iucC and iucD are involved in aerobactin synthesis and iutA is responsible for the expression of a specific outer membrane receptor protein for ferric aerobactin. Knockout mutants of iucD and iucDiutA in the APEC O2 strain E058 were constructed and named E058ΔiucD and E058ΔiucDΔiutA, respectively. To evaluate the pathogenicity of these mutants, we used multiple approaches to assess the effects of mutations on the virulence of APEC E058. Aerobactin-defective mutants E058ΔiucD and E058ΔiucDΔiutA showed significantly decreased pathogenicity compared with the wild-type strain E058, evidenced by the low extent of colonization in selected organs or being outcompeted by E058 in vivo. Chickens challenged with APEC E058 exhibited typical signs and lesions of avian colibacillosis, while those inoculated with the mutants E058ΔiucD or E058ΔiucDΔiutA showed moderate airsacculitis, mild pericarditis and perihepatitis. However, no significant differences in resistance to specific-pathogen-free chicken serum, ingestion by HD-11 cells, and growth rates in LB were observed between the mutants and the wild-type strain. These results indicated that the IucD- and IutA-related virulence factors play a significant role in the pathogenesis of the APEC strain E058., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Vorinostat (suberoylanilide hydroxamic acid) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. Intracellular access of vorinostat is essential to exert its epigenetic effects. Therefore, we studied the relationship between vorinostat extracellular (plasma) and intracellular (peripheral blood mononuclear cells, PBMCs) concentration and assessed its concentration-effect relationship by HDAC activity testing. Assays were developed and validated for the low nanomolar quantification of vorinostat and two inactive metabolites in human plasma and PBMCs. For the vorinostat extraction from plasma and PBMCs solid-phase extraction and liquid-liquid extraction methods were applied. Extraction recoveries ranged from 88.6% to 114.4% for all analytes and extraction methods. Extracts were chromatographed on a Phenomenex Luna column isocratically (plasma) or by gradient (PBMCs) consisting of acidic ammonium acetate, acetonitrile, and methanol. The analytes were quantified using deuterated internal standards and positive electrospray tandem mass spectrometry (multiple reaction monitoring) with lower limits of quantification of 11.0 ng/mL (plasma) and 0.1 ng/3 × 10(6) cells (PBMCs). The calibrated ranges were linear for vorinostat in plasma 11.0-1100 (11,000) ng/mL (metabolites) and PBMCs 0.1-10.0 ng/3 × 10(6) cells with correlation coefficients >0.99, an overall accuracy varying between -6.7% and +3.8% in plasma, -8.1% and -1.5% in PBMCs, and an overall precision ranging from 3.2% to 6.1% in plasma and 0.8% to 4.0% in PBMCs (SD batch-to-batch). The application to blood samples from healthy volunteers incubated with vorinostat revealed accumulation of vorinostat in PBMCs, effective intracellular HDAC inhibition at therapeutic vorinostat concentrations and a direct vorinostat concentration dependency to HDAC inhibition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The histone deacetylase inhibitor belinostat is being evaluated clinically as a single agent in the treatment of peripheral T-cell lymphomas and in combination with other anticancer agents to treat a wide range of human cancers including acute leukemias and solid tumors. To determine the pharmacokinetics of belinostat in the NCI ODWG liver dysfunction study, we developed and validated an LC-MS/MS assay for the quantitation of belinostat and five major metabolites in 0.05 mL human plasma. After protein precipitation, chromatographic separation was achieved with a Waters Acquity BEH C18 column and a linear gradient of 0.1% formic acid in acetonitrile and water. Detection with an ABI 4000Q mass spectrometer utilized both electrospray positive and negative mode ionization. The assay was linear from 30 to 5000 ng/mL for all six analytes and proved to be accurate (92.0-104.4%) and precise (CV <13.7%), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring parent drug and five major metabolites in plasma from a patient who was administered belinostat IV at a dose of 400 mg/m(2). The LC-MS/MS assay that has been developed will be an essential tool to further define the metabolism and pharmacology of belinostat in the ongoing liver organ dysfunction as well as other studies that investigate belinostat with other anticancer agents., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Fungal siderophores are likely to possess atheroprotective effects in humans, and therefore studies are needed to develop siderophore-rich food additives or functional foods to increase the siderophore uptake in people prone to cardiovascular diseases. In this study the siderophore contents of mould-ripened cheeses and meat products were analysed and the coprogen production by Penicillium nalgiovense was characterised., Results: High concentrations of hexadentate fungal siderophores were detected in penicillia-ripened Camembert- and Roquefort-type cheeses and also in some sausages. In one sausage fermented by P. nalgiovense, the siderophore content was comparable to those found in cheeses. Penicillium nalgiovense produced high concentrations of coprogen in submerged cultures, which were affected predominantly by the available carbon and nitrogen sources under iron starvation. Considerable coprogen yields were still detectable in the presence of iron when the fermentation medium was supplemented with the iron chelator Na₂-EDTA or when P. nalgiovense was co-cultivated with Saccharomyces cerevisiae., Conclusion: These data may be exploitable in the future development of high-siderophore-content foods and/or food additives. Nevertheless, the use of P. nalgiovense fermentation broths for these purposes may be limited by the instability of coprogen in fermentation media and by the β-lactam production by the fungus., (© 2012 Society of Chemical Industry.)

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila. An acetate kinase which can only utilize PP(i) but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus. In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD(+) by the enzymes pyruvate kinase and lactate dehydrogenase, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP(+) to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase. Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP(i).

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats., (Copyright © 2011. Published by Elsevier B.V.)

An ultra high performance liquid chromatography tandem mass spectrometry method (UHPLC-MS/MS) was developed and validated for the quantitation of LBH589, a novel histone deacetylase inhibitor (HDACi), in mouse plasma and tissues (liver, spleen, kidney and lung). Tobramycin was employed as the internal standard. Separation was performed on an Acquity UPLC™ BEH column, with a mobile phase consisting of 10% water (with 0.1% of trifluoroacetic acid) and 90% methanol (with 0.1% trifluoroacetic acid). LBH589 and tobramycin were determined using an electrospray ionization (ESI) interface. Detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring of the transitions of LBH589 at m/z 349.42→157.95 and of tobramycin at 468.2→163. Calibration curves for the UHPLC method (0.0025-1 μg/mL for plasma and tissue homogenates, equivalent to 0.0357-14.2857 μg/g for tissue samples) showed a linear range of detector responses (r>0.998). Intra-batch and inter-batch precision expressed as coefficient of variation (CV) ranged from 0.92 to 8.40%. Accuracy expressed as bias, ranged from -2.41 to 2.62%. The lower limit of quantitation (LLOQ) was 0.0025 μg/mL for both plasma and tissue homogenate samples, equivalent to 0.0357 μg/g tissue. This method was successfully applied to quantify LBH589 in plasma and tissue samples obtained after the intraperitoneal administration of a single dose of 20 mg/kg of LBH589 in BALB/c mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

New monolithic HPLC columns were prepared by γ-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 μm I.D.) and nano (100-75 μm I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R&#95;IC(50) and an averaged acetylation degree., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

Despite indications that S. granulatus and S. luteus release iron-chelating compounds, the exact spectrum of ferric hydroxamates synthesized by these two Suillus species remained unclear. Hence the aim of this study was to identify all of the main siderophores produced by these two ectomycorrhizal fungal species under pure culture conditions. By means of HPLC and LC-MS analyses we show that S. granulatus releases cyclic and linear fusigen, ferrichrome, coprogen and triacetylfusarinine C into the nutrient medium, while S. luteus culture filtrates contain cyclic and linear fusigen, ferricrocin and coprogen. All of the different siderophores were identified on basis of reference compounds and their specific MS spectra which were recorded on a high resolution MS in positive electrospray ionisation mode. Initial HPLC separations were performed on a C-18 stationary phase, using an acidic eluent (0.1% formic acid in water and acetonitrile) in gradient mode. The potential of these two ectomycorrhizal fungal species to produce siderophores representing three different groups of hydroxamates is discussed in relation to its ecological significance.

Scedosporium apiospermum is an emerging pathogen colonizing the airways of patients with cystic fibrosis (CF). While usually responsible for chronic colonization without clinical signs, this fungus may cause severe and often lethal infections in lung transplant recipients. Early diagnosis of its airway colonization and appropriate treatment are required to eradicate the fungus when a lung transplantation is planned. Here we propose an alternative to mycological examination of sputum samples based on extraction of siderophores by chromatography on Amberlite XAD-4, followed by high performance liquid chromatography analysis of the siderophore extract. Improvement of the extraction procedure was performed in a fractional factorial design which revealed the importance of prior ammonium sulfate precipitation of the proteins, alkalinization of the obtained solution and stirring during extraction. In order to verify the specificity of N(α)-methyl coprogen B for S. apiospermum, the method was applied on culture supernatants of different filamentous fungi colonizing the airways of CF patients, including some aspergilli and Exophiala dermatitidis. N(α)-methyl coprogen B was detected exclusively for species of the S. apiospermum complex. Likewise, sputum samples from colonized and non-colonized CF patients were analyzed, and the siderophore was detected exclusively in three out of the five specimens which were found by culture to contain S. apiospermum. Together these results confirmed N(α)-methyl coprogen B as a marker of the airway colonization by species of the S. apiospermum complex.

The biochemical and molecular mechanisms used by alkaliphilic bacteria to acquire iron are unknown. We demonstrate that alkaliphilic (pH > 9) Bacillus species are sensitive to artificial iron (Fe³+) chelators and produce iron-chelating molecules. These alkaliphilic siderophores contain catechol and hydroxamate moieties, and their synthesis is stimulated by manganese(II) salts and suppressed by FeCl₃ addition. Purification and mass spectrometric characterization of the siderophore produced by Caldalkalibacillus thermarum failed to identify any matches to previously observed fragmentation spectra of known siderophores, suggesting a novel structure.

A novel, sensitive and reliable liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the determination of belinostat (PXD101) in human plasma. Oxamflatin was used as the internal standard. Liquid-liquid extraction of the plasma sample was performed using tert-butyl methyl ether as the organic solvent. Chromatographic separation was achieved on a BDS Hypersil C18 column (2.1 mm x1 00 mm, 5 microm) using gradient elution mode using 0.05% formic acid in water and 0.05% formic acid in acetonitrile as solvents A and B, respectively, 60/40. The run time was 6 min. The mass spectrometer was operated under a positive electrospray ionization condition and a multiple reaction monitoring mode. An excellent linear calibration was achieved in the range of 0.5-1000 ng/mL. An average recovery of belinostat for four quality controls was 72.6% and the recovery of the internal standard at 1000 ng/mL was 67.8%. The intra-day and inter-day precisions for belinostat were

Background and Objectives: The aim of this study was to determine the role of host factors and bacterial virulence genes in the development of pyelonephritis caused by Escherichia coli in renal transplant (Tx) recipients., Design, Setting, Participants, & Measurements: A total of 328 E. coli isolates from cases of cystitis (Cys; n=239) or pyelonephritis (PN; n=89), with 169 from renal Tx recipients, were subjected to molecular analyses to identify P-fimbria subunits (PapC, PapG II, and PapGIII), G- and M-fimbriae, and aerobactin. The presence of antibiotic resistance was also determined. Parameters such as gender, age, immunosuppression regimens, causes of ESRD, kidney donor, intraoperative anastomosis, use of double J stent, trimethoprim/sulfamethoxazole (TMP/SMZ) prophylaxis, and time after Tx were evaluated., Results: A multivariate analysis showed a significant association between PN and renal Tx. In renal Tx recipients, the risk of occurrence of PN was significantly higher among males and for those no longer receiving TMP/SMZ prophylaxis. E. coli strains isolated from PN presented a lower prevalence of papGIII and lower rates of resistance to pipemidic acid. Although papGII was more prevalent in PN than in Cys, it was not independently associated with PN., Conclusions: These findings suggested that renal Tx increases the risk for PN, and the male sex represented a host factor independently associated with risk, whereas the prophylaxis with TMP/SMZ was protective. The lack of papGIII and low resistance to first-generation quinolones were bacterial-independent risk factors for PN in Tx.

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.

Modafinil, adrafinil and their related substances were synthesized and analyzed by RP-LC with ESI-MS/MS. The ionization mode, polarity, cone voltage, and chromatographic conditions were evaluated. The optimum LC-MS conditions to obtain fragment ions indispensable for identification of the structures were described. The bulk drugs purity of modafinil and adrafinil was evaluated on Kromasil C(18) column with ACN/0.02 M ammonium acetate as mobile phase in gradient elution mode at 30 degrees C. The method was found to be suitable not only for monitoring the reactions during the process development but also for quality assurance of modafinil and adrafinil.

A highly sensitive and specific liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for investigating the pharmacokinetics of adrafinil in rats was developed. Rat serum pretreated by solid-phase extraction (SPE) was analyzed by LC-MS/MS with an electrospray ionization (ESI) interface. The mobile phase consisted of acetonitrile:water:acetic acid (35:65:0.1, v/v/v) in an isocratic elution mode pumped at 1.0 ml/min. The analytical column (250 mm x 4.6 mm i.d.) was packed with Kromasil C(18) material (5.0 microm). The standard curve was linear from 16.5 to 5000 ng/ml. The assay was specific, accurate (R.S.D.<2.6%), precise and reproducible (within- and between-day precisions R.S.D. <7.0% and <9.0%, respectively). Adrafinil in rat serum was stable over three freeze-thaw cycles at ambient temperature for 6 h. The method had a lower limit of quantitation of 16.5 ng/ml, which offered high sensitivity for the determination of adrafinil in serum. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by tandem mass spectrometry is described for the simultaneous quantitative determination of decitabine (5-aza-2'-deoxycytidine) and vorinostat (Suberoylanalide hydroxamic acid, SAHA) in human plasma. The method involves a simple acetonitrile precipitation step and centrifugation followed by injection of the supernatant onto a C18 150mmx2.1mm I.D., 3microm HPLC column at 36 degrees C. Separation of decitabine, SAHA and their respective internal standards was achieved with a gradient elution and detection was via the mass spectrometer operated in selected reaction monitoring mode. The method was within the defined validation parameters for linearity, repeatability, reproducibility and stability. The limit of detection was determined as 1.0 and 0.125ngml(-1) and lower limits of quantitation were 10 and 1ngml(-1) for decitabine and SAHA, respectively. Effects of sample preparation on stability were also evaluated in human plasma. For clinical sample handling tetrahydrouridine, an inhibitor of cytidine deaminase was found to help prevent decitabine degradation. The method is currently being used in clinical pharmacokinetic studies for the evaluation of decitabine and SAHA combination therapies.

Wheat samples were taken at different stages of germination characterized by their falling number (which is a relevant indicator of germination) from 400 to 60 s. Each batch was treated by the Oxygreen process, a treatment by ozone, in a closed sequential batch reactor. Leucotriene B4 (LTB 4) was induced by germination, but ozone treatment did not increase this effect. Extract obtained from these wheat batches was applied on human epithelial bronchial cells. Wheat extract from nongerminated wheat did not induce any DNA adduct. More the wheat germination gets underway, more DNA adducts are observed. In contrast, germination did not affect the cell viability. Ozonization of wheat exemplified genotoxic effects only if the wheat was germinated. The implication of hydroxamic acids is discussed. In conclusion, ozonization of wheat, of high milling quality, does not pose any problem.

Clinical isolates of enterohaemorrhagic Escherichia coli, both O157 and non-O157 serotypes, were investigated for siderophore production, for growth promotion by haem and esculetin in iron-restricted conditions, for production of enterohaemolysin and esculin hydrolase, and for the presence of the chuA and ehx genes by PCR. As expected, all the strains produced enterobactin, but the prevalence of other factors varied among the serovars tested. None of the O157 and O26 strains produced aerobactin or "colibactin", whereas among other enterohaemorrhagic E. coli non-O157 serovars the frequencies of aerobactin and "colibactin" production were similar to those of commensal E. coli strains. The ability to use ferric esculetin for growth in iron-limited media was markedly more prevalent among non-O157 serovars and less prevalent among O157 strains compared with commensal E. coli strains. Almost all O157, O26 and O103 strains expressed enterohaemolysin, compared with only 50% of other non-O157 strains. Similarly, almost all O157 and O26 strains utilised haem as a host iron source; the frequency of haem use by other non-O157 strains was generally lower and variable among serovars, such that none of the O103:H2 isolates tested used haem as an iron source. The gene chuA, which encodes the haem transport protein ChuA and which is prevalent in O157:H7 strains, was only rarely noted among non-O157 serovars of enterohaemorrhagic E. coli, even among isolates that could use haem as an iron source. Overall our data demonstrate that O157:H7 and non-O157 serovars, in particular O26:H(-)/H11 and O103:H2, use distinctly different strategies for obtaining iron, and suggest two evolutionary distinct lines of enterhaemorrhagic E. coli.

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.

Aims: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo., Methods and Results: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae., Conclusions: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans., Significance and Impact of the Study: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.

Benzoxazinones are naturally occurring secondary metabolites of some Gramineae plants, responsible for their resistance to some pathogenic fungi and for their allelopathic action. Six varieties of winter wheat grown in fields under organic or conventional systems and 11 old accessions were tested for two consecutive seasons and three plant development stages for the concentration in their roots of cyclic hydroxamic acids and their degradation products. This is the first report of six benzoxazinones analyzed in plants grown in the field. An analytical technique employing LC-DAD was used for determination. It was shown that 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, its degradation product 6-methoxybenzoxazolin-2-one, and the lactam 2-hydroxy-7-methoxy-1,4-benzoxazin-2-one were predominant compounds in all tested samples. Their concentrations significantly differed with plant development stage and season, but no significant differences were found between varieties and between plant cultivation systems. The concentrations of 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its degradation product benzoxazolin-2-one (BOA) were much lower, ranging from 60 to 430 mg/kg of dry matter, depending on accession, stage of development, and season. There was no significant difference found between plants grown in different cultivation systems, but there were significant differences between old and new varieties; concentrations of DIBOA and its derivatives were significantly lower in old accessions. It was concluded that the concentrations of DIBOA and BOA, which are precursors of highly fungicidal 2-aminophenol, 2-amino-3H-phenoxazin-3-one, and 2-acetylamino-3H-phenoxazin-3-one, are theoretically high enough to protect plants against some soilborne pathogens.

Mechanisms of fragmentation of hydroxamic siderophores are proposed comparing deuterated and nondeuterated samples. Standard siderophores (e.g. deferoxamine and coprogen) were directly injected into both ion trap and linear quadrupole mass spectrometers with electrospray ionization (ESI). Four and two fragmentation steps were carried out for deferoxamine and coprogen (analyzed by positive and negative ESI, respectively). Deferoxamine cleavages occurred in both peptide and hydroxamic bonds while the coprogen fragmentation pattern is more elaborate, since it contains Fe(III) in its structure.

NMR spectra (1H, 13C, 15N) of para- and meta-substituted benzohydroxamic acids were studied in dry dimethyl sulfoxide solutions. The 13C chemical shifts were very close to those found by cross-polarization magic angle spinning in solids, the hydroxamic (not hydroximic) structure of which is unambiguous. The hydroxamic structure of these acids in DMSO solutions was proved independently by their 15N chemical shifts. The 15N and 1H chemical shifts of the NH-OH fragment showed excellent mutual dependences and dependences on the nature of the ring substituent. According to these dependences and ab initio energy calculations, all the acids assume the same Z conformation. Proton exchange between hydroxamic OH and NH groups in DMSO proceeded by both intra- and intermolecular exchange and the rates did not exhibit any simple relationship to the substituent constants., (Copyright 2005 John Wiley & Sons, Ltd)

Wheat (Triticum aestivum) is the main food crop in the world, and plays an important role in agricultural production. In order to enhance wheat yield, herbicides and germicides were intensively applied and made negative effects on the environment. Wheat possesses allelopathic potential for weed suppression and disease control through the release of secondary metabolites from its living plants or residues, which could avoid the environment pollution brought by herbicides and germicides. This paper reviewed the research advances in wheat allelopathy. Hydroxamic acids and phenolic acids are the predominant allelochemicals frequently reported which could produce plant natural defense against weed, pest and disease. The allelopathic activity of allelochemicals is determined not only by the allelochemicals, but also by the factors of inheritance, environment and biology. The retention, transportation and transformation processes of allelochemicals, and the relationship between wheat allelopathy and soil biota and its mechanism were seldom studied and still needed to be researched profoundly. Utilizing wheat allelopathy in plant protection, environment protection and crop breeding would improve the stress-resistance, yield and quality of wheat in agricultural production.

The research advance in cyclic hydroxamic acids was reviewed in this paper. Cyclic hydroxamic acids are the important natural products of cereal crops. They and their respective derivatives are the constitutive compounds of a wide variety of gramineous plants and few dicot plants. They have structural diversity and different natural occurrences. Because of their phytotoxic properties, cyclic hydroxamic acids show a great variety of biological activities. They are the defensive agents against plant diseases, pests, nematodes and other plants. The distribution of cyclic hydroxamic acids in Zea mays and their variation in relation to the age were focused on in the paper. In Zea mays, there are structural diversity of cyclic hydroxamic acids and related benzoxazolinones. DIMBOA (1,4-benzoxazin-3(4H)-ones) is the most abundant derivative in Zea mays. The content of cyclic hydroxamic acids is strongly cultivar-dependent in Zea mays. Hydroxamic acids are not present in seeds. After germination, the level of DIMBOA increases, and the maximum level occurs in young seedlings a few days after germination. DIMBOA exists in all parts of plants, and its concentration is generally higher in shoots than in roots. In all stages, the young leaves of Zea mays have relatively high content of DIMBOA. The concentrations of these hydroxamic acids are highly dependent on environmental growth conditions. Under UV-light and water deficiencies, the levels of hydroxamic acids in plant increase rapidly. Cyclic hydroxamic acids exuded by Zea mays root can be quantitatively analyzed by HPLC. Supplying iron can significantly increase the exudation of DIMBOA from Zea mays root.

A soluble N-terminal domain of the Escherichia coli adenylyl transferase (ATase) is responsible for deadenylylation activity of the intact enzyme. Previous studies of the deadenylylation activity have involved a fragment, AT-N423 (residues 1 to 423), which was extended by 17 amino acids to give AT-N440. This new domain is truncated at the end of a predicted helix and prior to a Q-linker. The domain was found to be very soluble and stable so that it could be purified to homogeneity and crystallized. This construct has deadenylylation activity that is independent of the low nitrogen status indicator PII-UMP. The crystals belong to space group P3(1)21 or its enantiomorph P3(2)21 with a=b=116.6 A and c=67.6 A.

The aqueous solution reactions of the title compound, 1, were examined for comparison to those previously reported for another model carcinogen N-pivaloyloxy-2-amino-alpha-carboline, 2. Both of these are models for the ultimate carcinogenic metabolites of 2-amino-alpha-carboline (AalphaC), a food-derived heterocyclic amine mutagen and carcinogen. The present study was undertaken to determine the effect of the N-acetyl group on the chemistry of such compounds. The N-acetyl group slows down N-O bond cleavage by a factor of (5.5 x 10(3))-fold. This allows other reactions not observed in 2, or in other model carcinogens, to be observed. Among these are acyl-transfer reactions to the aqueous solvent, both uncatalyzed and catalyzed by N3-. In addition, the conjugate acid of 1, 1H+, is subject to a spontaneous decomposition not previously observed in other esters of heterocyclic hydroxylamines or hydroxamic acids. This reaction yields the hydroxylamine, 5, and does so without the intermediacy of the hydroxamic acid, 3, and with 18O exchange from the solvent into the hydroxylamine O. This unique reaction may be caused by an intramolecular proton donation by the pyridyl N-H to the amide carboxyl that catalyzes an intramolecular nucleophilic attack by the carboxyl O of 1H+. A nitrenium ion pathway can still be detected for 1, but, unlike 2 and related esters, this reaction is in competition with other processes throughout the pH range of the study.

In this study a new approach to determine three different siderophores (ferrichrome, ferrichrysin, ferricrocin) in natural soil solutions as well as in cultures of fungi is presented. The method includes enrichment of the analytes on a short pre-column, packed with C18 material, and subsequent highly selective separation of the analytes on a capillary porous graphitic carbon (PGC) column. In contrast to normal C18 packing materials, porous graphitic carbon offers chromatographic resolution between the three very similar analytes. The selectivity of the method is enhanced even further by the electrospray ionization (ESI) mass spectrometric detection. The combination of a short pre-column and a packed capillary separation column results in a method with high sensitivity. Reported detection limits, defined as the concentration giving the signal-to-noise ratio 3:1, is 27.7 pM for ferrichrome, 46.1 pM for ferricrocin and 37.4 pM for ferrichrysin.

Systematic chemical genetics aims to explore the space representing interactions between small molecules and biological systems. Beyond measuring binding interactions and enzyme inhibition, measuring changes in the activity of proteins in intact signaling networks is necessary. Toward this end, we are partitioning chemical space into regions with different biological activities using a panel of cell-based assays and small molecule "chemical genetic modifiers." Herein, we report on the use of this methodology for the discovery of 617 small molecule inhibitors of histone deacetylases from a multidimensional screen of an encoded, diversity-oriented synthesis library. Following decoding of chemical tags and resynthesis, we demonstrate the selectivity of one inhibitory molecule (tubacin) toward alpha-tubulin deacetylation and another (histacin) toward histone deacetylation. These small molecules will facilitate dissecting the role of acetylation in a variety of cell biological processes.

Co-extraction of lipid materials is the major source of interference in determinations of low-polarity compounds in many biological matrixes. "SFE-plus-C(18)", a recently developed supercritical fluid extraction method employing C(18) adsorbent in the extraction chamber, can enable selective extraction of low-polarity compounds in lipid-rich biological matrixes without a cleanup step. This study reports the application of the SFE-plus-C(18) method to the quantification of: 1. polycyclic aromatic hydrocarbons (PAH) in commercially purchased smoked fish; and 2. anti-cancer agents cyclophosphamide (CP) and suberoylanilide hydroxamic acid (SAHA) spiked into homogenized whole bovine milk. Over the course of SFE-plus-C(18)extraction, indigenous lipids are preferentially retained on the C(18) adsorbent. Compared with the conventional method, only 8-15% of the lipids in the smoked fish sample, and only 6-18% of the lipids in the milk sample, were co-extracted by SFE-plus-C(18). This reduction in the quantity of background lipids significantly improved chromatographic separations, retarded deterioration of the column, and dramatically improved the ability to quantify PAH present at trace levels in smoked fish by GC-MS. Using the SFE-plus-C(18) method, ten targeted PAH were detected in the range 9.5-13.5 ng g(-1) in the smoked fish sample. Compared with these levels, PAH extractions by use of conventional SFE gave values that were lower by 38-86%. Recoveries of CP and SAHA spiked into milk were close to 100% in both SFE-plus-C(18)and conventional SFE, where the lipid background during the chromatographic elution of CP and SAHA was not so severe.

Hydroxamic acids are widespread in the tissues of plants, in metabolites of bacteria and fungi, including complex compounds with metal ions. These acids have wide spectrum of biological activity and therefore are perspective reagents for analysis of chemical elements. Fourteen aliphatic and aromatic derivatives of hydroxamic acids have been synthesized from esters of carboxylic acids. Photometric reactions of hydroxamic acids with iron (II) and (III) were investigated. Complex formation of iron (II) and (III) depending on pH was studied with series of synthesized hydroxamic acids: octanohydroxamic, maleic hydroxamic, 2-hydroxybenzoxydroxamic, benzoxydroxamic, phthalmonoxydroxamic and 3-metoxybenzohydroxamic acids. Composition of iron (III) complexes with 2-hydroxybenzohydroxamic, octanoxydroxamic, 3-metoxybenzohydroxamic acids and iron (II) with 2-hydroxybenzohydroxamic acid was studied by methods of mole ratio and isomolar solutions. Sensitivity of reagents was evaluated by values of absorption coefficients (epsilon). Stability of complexes in water and organic solvents was investigated. Interaction between iron (III) and hydroxamic acids (octanoxydroxamic, 2-hydroxybenzohydroxamic, 3-metoxybenzohydroxamic) have been applied for quantitative photometric analysis of iron (III) salts. Color reaction of iron (II) with 2-hydroxybenzohydroxamic acid was applied for quantitative photometric determination of iron (II) salts. 3-Metoxybenzohydroxamic acid was proposed as a new indicator for complexonometric analysis of iron (III). Chelatometric titration of iron (III) using this indicator is not influenced by copper, cobalt, zinc, manganese, so this methods is recommended for iron quantity detection in antianemic drugs, which are composed of latter microelements. Synthesis procedure of 2-benzoylamino-3-arylacrylhydroxamic acids from saturated azlactones was created. Color and precipitate reactions of iron (II) and (III), copper (II), nickel (II) and cobalt (II) ions with four newly synthesized acids (with and without substitutes in aromatic ring) were studied. Sensitivities of reactions between 2-benzoylamino-3-arylacrylhydroxamic acids and iron (II) and (III) were evaluated and compared with 2-hydroxybenzohydroxamic acid.

The incidence of bacteriocinogeny and lysogeny was followed in bacteria of 3 recently acknowledged species of the genus Escherichia: E. hermanii, E. vulneris and E. fergusonii. Almost all of the strains examined were of human origin. In 30 strains of E. hermanii no one was found bacteriocinogenic while 57% were lysogenic, in 30 strains of E. vulneris none was found to be bacteriocinogenic and only 10% were lysogenic, and in 50 strains E. fergusonii 12% were bacteriocinogenic and 40% lysogenic. From the 6 bacteriocinogenic strains of E. fergusonii, 3 were producers of colicin E1, 2 of colicin Ib and 1 of colicin Ia. In addition, 3 E. fergusonii strains produced aerobactin.

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied. Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum. These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality. Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.

In eleven strains of genus Enterococcus the growth kinetics and hydroxamate siderophore production in iron-deficient medium were investigated. The classical pattern of bacterial growth kinetics and siderophore synthesis was observed.

Hydroxamic acid content was analyzed in the root tissue of four maize, Zea mays L., lines using high-performance liquid chromatography (HPLC) and related to western corn rootworm, Diabrotica virgifera virgifera LeConte, larval development and survivorship. Maize lines evaluated included Mp710 (PI 596627), MpSWCB-4, (PI 550498), Sc213 (PI 548792), and Dk580 (DeKalb commercial hybrid). Maize plants from each line were grown in test tubes containing a transparent agarose gel medium in a growth chamber. After 8 d of growth, root tissue of each line was harvested and hydroxamic acid content analyzed using HPLC. Three hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), 6-methoxybenzoxazolinone (MBOA), and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), were identified in the maize roots tested. DIMBOA concentration was quantified and ranged from 246.37 +/- 70.53 micrograms to 91.84 +/- 49.82 micrograms DIMBOA per gram of root tissue. No significant difference was found among lines in D. v. virgifera larval development and survivorship.

LC-NMR and LC-MS were applied to the characterization of six degradation products of a protease inhibitor, N-hydroxy-1,3-di-[4-ethoxybenzenesulphonyl]-5,5-dimethyl-[1,3]c yclohexyldiazine-2-carboxamide, in a dosage formulation. A reversed-phase HPLC method was developed for the separation of the parent compound and its six degradation products. LC-MS was then utilized to obtain the molecular weight and fragmentation information using an electrospray ionization (ESI) interface in the positive ion mode. LC-NMR was employed to acquire detailed structural information using a selective solvent suppression pulse sequence in the stop flow mode. This work demonstrated the usefulness of this integrated approach for the rapid and unambiguous identification of drug compounds and their degradation products in dosage formulations.

An extracellular low-molecular mass component (LMMC) with catalytic properties was isolated from liquid cultures containing wheat bran of ascomycete thermophilic Thermoascus aurantiacus. The partially purified LMMC showed very high activity with typical phenoloxidase substrates in the absence of hydrogen peroxide at acidic pH (2.8). However, in this pH range, the phenoloxidase (PO) activity was quickly lost. The LMMC showed a high optimum temperature (80 degrees C) and an elevated thermostability. The molecular mass of the component estimated by gel filtration chromatography was 530 Da. IR and 1H- and 13C-NMR spectra indicated the presence of hydroxamic acid moiety. Qualitative determination of metal ions by several techniques revealed the presence of mainly iron associated with this structure. Iron may be the responsible for the ability for catalyze oxidation reactions, such as o-dianisidine oxidation, by the LMMC. These results suggested the existence of a hydroxamate-type metal-binding component, most likely hydroxamate siderophore. In addition, the chrome azurol S (CAS) universal assay for noncomplexed siderophores detection revealed the production of these compounds by T.aurantiacus in solid and liquid media., (Copyright 1999 Academic Press.)

Thirty one Escherichia coli strains isolated from pigs with urinary tract infections were investigated for presence of virulence factors and plasmid DNA profile. The most frequent virulence factors presented by these strains were mannose-resistant fimbriae, including P. fimbriae (54.8%) and aerobactin production (45.2%). The pap) operon, detected by PCR, was found in 54.8% of the strains, which is similar to its frequency in human strains. Other characteristics such as the presence of mannose-sensitive hemagglutinin (16.1%), indicative of type 1 pili, and production of hemolysin (25.8%), colicin (38.7%) and toxins (22.6% for LT and for VT) were less frequent. No strains were positive for STa production. Plasmid profiles were variable among isolates from either the same or different farms.