Blood culture-based diagnosis of bacteraemia: state of the art.

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

Conjugation of a Ru(II) Arene Complex to Neomycin or to Guanidinoneomycin Leads to Compounds with Differential Cytotoxicities and Accumulation between Cancer and Normal Cells

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η6-p-cym)RuCl(PPh3)2]+, allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N′-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 μM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 μM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 μM in DU-145 and IC50 = 11.33 μM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 μM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 μM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

(MA)LDI MS Imaging at High Specificity and Sensitivity

(Matrix-assisted) laser desorption/ionization ((MA)LDI) mass spectrometry imaging (MSI) has been driven by remarkable technological developments in the last couple of years. Although molecular information of a wide range of molecules including peptides, lipids, metabolites, and xenobiotics can be mapped, (MA)LDI MSI only leads to the detection of the most abundant soluble molecules in the cells and, consequently, does not provide access to the least expressed species, which can be very informative in the scope of disease research. Within a short period of time, numerous protocols and concepts have been developed and introduced in order to increase MSI sensitivity, including in situ tissue chemistry and solvent-free matrix depositions. In this chapter, we will discuss some of the latest developments in the field of high-sensitivity MSI using solvent-free matrix depositions and will detail protocols of two methods with their capability of enriching molecular MSI signal as demonstrated within our laboratory.

Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

Sintesis de poliuretanos a partir de polioles obtenidos a partir del aceite de higuerilla modificado por transesterificación con pentaeritritol

Castor oil was reacted by transesterification with various percentages in mass of pentaerythritol to obtain different esters of pentaerythritol. Alternatively, glycerol was also used instead of pentaerythritol for the same reaction in order to establish comparative reference products. The products of the reactions were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in order to detect and quantify (in terms of the molecular mass and structural information) the components of the products obtained. Analysis for hydroxyl value, acid value, viscosity and specific gravity were used to complete the characterization of the polyols obtained and also of the original castor oil. The polymer characterization was accomplished by tensile stress-strain tests, Shore A hardness, thermogravimetric analysis and chemical resistance to solvents.

cDNA and deduced primary structure of basic phospholipase A2 with neurotoxic activity from the venom secretion of the Crotalus durissus collilineatus rattlesnake

To illustrate the construction of precursor complementary DNAs, we isolated mRNAs from whole venom samples. After reverse transcription polymerase chain reaction (RT-PCR), we amplified the cDNA coding for a neurotoxic protein, phospholipase A2 D49 (PLA2 D49), from the venom of Crotalus durissus collilineatus (Cdc PLA2). The cDNA encoding Cdc PLA2 from whole venom was sequenced. The deduced amino acid sequence of this cDNA has high overall sequence identity with the group II PLA2 protein family. Cdc PLA2 has 14 cysteine residues capable of forming seven disulfide bonds that characterize this group of PLA2 enzymes. Cdc PLA2 was isolated using conventional Sephadex G75 column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The molecular mass was estimated using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. We tested the neuromuscular blocking activities on chick biventer cervicis neuromuscular tissue. Phylogenetic analysis of Cdc PLA2 showed the existence of two lines of N6-PLA2, denominated F24 and S24. Apparently, the sequences of the New World’s N6-F24-PLA2 are similar to those of the agkistrodotoxin from the Asian genus Gloydius. The sequences of N6-S24-PLA2 are similar to the sequence of trimucrotoxin from the genus Protobothrops, found in the Old World.

Proteomic analysis of cytosolic proteins associated with petite mutations in Candida glabrata

The incidence of superficial or deep-seated infections due to Candida glabrata has increased markedly, probably because of the low intrinsic susceptibility of this microorganism to azole antifungals and its relatively high propensity to acquire azole resistance. To determine changes in the C. glabrata proteome associated with petite mutations, cytosolic extracts from an azole-resistant petite mutant of C. glabrata induced by exposure to ethidium bromide, and from its azole-susceptible parent isolate were compared by two-dimensional polyacrylamide gel electrophoresis. Proteins of interest were identified by peptide mass fingerprinting or sequence tagging using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer. Tryptic peptides from a total of 160 Coomassie-positive spots were analyzed for each strain. Sixty-five different proteins were identified in the cytosolic extracts of the parent strain and 58 in the petite mutant. Among the proteins identified, 10 were higher in the mutant strain, whereas 23 were lower compared to the parent strain. The results revealed a significant decrease in the enzymes associated with the metabolic rate of mutant cells such as aconitase, transaldolase, and pyruvate kinase, and changes in the levels of specific heat shock proteins. Moreover, transketolase, aconitase and catalase activity measurements decreased significantly in the ethidium bromide-induced petite mutant. These data may be useful for designing experiments to obtain a better understanding of the nuclear response to impairment of mitochondrial function associated with this mutation in C. glabrata.

Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

Cyclotetrasiloxane Frameworks for the Chemoenzymatic Synthesis of Oligoesters

Immobilized lipase B from Candida antarctica (Novozym® 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65– 80% consumption of starting materials in 24–48 h. Longer reaction times, 72–96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw ¼ 11 400, 13 100, and 19 400 g mol 1 for the substrate pairs of C7D4 ester/ octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw ¼ 86 800 g mol 1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.

Lactide polymerization with iron alkoxide complexes

Les essais préliminaires pour préparer des alcoolates de fer à partir du bichlorure ou bibromure de fer (II), en les combinant avec des ligands de type diimino pyridine, ont engendré la formation de complexes homoleptiques et hétéroleptiques, dépendant des substituants sur les branches imines du ligand. Ces complexes homoleptiques octaédriques et paramagnétiques ont été étudiés par rapport à leurs propriétés spectroscopiques et cristallographiques. De plus, la synthèse des complexes de fer hétéroleptique a engendré de bons précurseurs penta-coordonnés pour les réactions de substitution de ligands avec des alcoolates de métaux alcalins, de manière à produire les dialcoolates de fer (II) désirés. Des techniques d’analyse telles que la spectroscopie UV-vis, l’analyse élémentaire, la spectrométrie de masse à haute résolution et la cristallographie aux rayons X ont été utilisées pour caractériser ces complexes de fer. L’activité catalytique de ces complexes de fer (II) a aussi été étudiée par rapport à la polymérisation du lactide; les dialcoolates convoités ont été générés in-situ en raison de la difficulté à produire et à isoler les dérivés alcoolates des complexes diimino pyridine de fer. Une étude approfondie a aussi été faite sur les réactions de polymérisation, surtout par rapport aux valeurs de conversion à l’échelle du temps, ainsi qu’à la tacticité des chaines de polymères obtenues. Ces analyses ont été effectuées par l’entremise de la spectroscopie de résonance magnétique nucléaire, de la chromatographie d’exclusion stérique, et de la spectrométrie de masse MALDI (désorption-ionisation laser assistée par matrice).

Characterization of an extracellular alkaline serine protease from marine Engyodontium album BTMFS10

An alkaline protease from marine Engyodontium album was characterized for its physicochemical properties towards evaluation of its suitability for potential industrial applications. Molecular mass of the enzyme by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) analysis was calculated as 28.6 kDa. Isoelectric focusing yielded pI of 3–4. Enzyme inhibition by phenylmethylsulfonyl fluoride (PMSF) and aprotinin confirmed the serine protease nature of the enzyme.Km, Vmax, and Kcat of the enzyme were 4.727 9 10-2 mg/ml, 394.68 U, and 4.2175 9 10-2 s-1, respectively. Enzyme was noted to be active over a broad range of pH (6–12) and temperature (15–65 C), withmaximumactivity at pH 11 and 60 C. CaCl2 (1 mM), starch (1%), and sucrose (1%) imparted thermal stability at 65 C. Hg2?, Cu2?, Fe3?, Zn2?, Cd?, and Al3? inhibited enzyme activity, while 1 mMCo2? enhanced enzyme activity. Reducing agents enhanced enzyme activity at lower concentrations. The enzyme showed considerable storage stability, and retained its activity in the presence of hydrocarbons, natural oils, surfactants, and most of the organic solvents tested. Results indicate that the marine protease holds potential for use in the detergent industry and for varied applications.

UV Embossed Plastic Chip for Protein Separation and Identification

This report demonstrates a UV-embossed polymeric chip for protein separation and identification by Capillary Isoelectric Focusing (CIEF) and Matrix Assisted Laser Desportion/Ionization Mass Spectrometry (MALDI-MS). The polymeric chip has been fabricated by UV-embossing technique with high throughput; the issues in the fabrication have been addressed. In order to achieve high sensitivity of mass detection, five different types of UV curable polymer have been used as sample support to perform protein ionization in Mass Spectrometry (MS); the best results is compared to PMMA, which was the commonly used plastic chip for biomolecular separation. Experimental results show that signal from polyester is 12 times better than that of PMMA in terms of detection sensitivity. Finally, polyester chip is utilized to carry out CIEF to separate proteins, followed by MS identification.

Introduction of 4-chloro-alpha-cyanocinnamic acid liquid matrices for high sensitivity UV-MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) is a key ionization technique in mass spectrometry (MS) for the analysis of labile macromolecules. An important area of study and improvements in relation to MALDI and its application in high-sensitivity MS is that of matrix design and sample preparation. Recently, 4-chloro-alpha-cyanocinnamic acid (ClCCA) has been introduced as a new rationally designed matrix and reported to provide an improved analytical performance as demonstrated by an increase in sequence coverage of protein digests obtained by peptide mass mapping (PMM) (Jaskolla, T. W.; et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12200-12205). This new matrix shows the potential to be a superior alternative to the commonly used and highly successful alpha-cyano-4-hydroxycinnamic acid (CHCA). We have taken this design one step further by developing and optimizing an ionic liquid matrix (ILM) and liquid support matrix (LSM) using ClCCA as the principle chromophore and MALDI matrix compound. These new liquid matrices possess greater sample homogeneity and a simpler morphology. The data obtained from our studies show improved sequence coverage for BSA digests compared to the traditional CHCA crystalline matrix and for the ClCCA-containing ILM a similar performance to the ClCCA crystalline matrix down to 1 fmol of BSA digest prepared in a single MALDI sample droplet with current sensitivity levels in the attomole range. The LSMs show a high tolerance to contamination such as ammonium bicarbonate, a commonly used buffering agent.

Stress-induced changes in the Schizosaccharomyces pombe proteome using two-dimensional difference gel electrophoresis, mass spectrometry and a novel integrated robotics platform

Robotic and manual methods have been used to obtain identification of significantly changing proteins regulated when Schizosaccharomyces pombe is exposed to oxidative stress. Differently treated S. pombe cells were lysed, labelled with CyDye and analysed by two-dimensional difference gel electrophoresis. Gel images analysed off-line, using the DeCyder image analysis software [GE Healthcare, Amersham, UK] allowed selection of significantly regulated proteins. Proteins displaying differential expression were excised robotically for manual digestion and identified by matrix-assisted laser desorption/ionisation - mass spectrometry (MALDI-MS). Additionally the same set of proteins displaying differential expression were automatically cut and digested using a prototype robotic platform. Automated MALDI-MS, peak label assignment and database searching were utilised to identify as many proteins as possible. The results achieved by the robotic system were compared to manual methods. The identification of all significantly altered proteins provides an annotated peroxide stress-related proteome that can be used as a base resource against which other stress-induced proteomic changes can be compared.

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two-dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix-assisted laser-desorption ionisation time of flight mass spectrometry (MALDI - TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two-dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin-like proteins or glucanases, and cell expansion, i.e. β-D-glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.