MALDI-TOF Mass Spectrometry Analysis Research Articles

Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20–30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Identification of the active protein in rice bran protein having an inhibitory activity of cholesterol micellar solubility.

In our previous study, rice bran protein (RBP) inhibited cholesterol micellar solubility in vitro and decreased serum cholesterol level in rats. In the present study, RBP was separated and purified by size-exclusion chromatography and reversed-phase chromatography. The active protein of RBP related to cholesterol micellar solubility was identified as lectin and non-specific lipid-transfer protein 1 using MALDI-TOF mass spectrometry analysis.

Enhanced expression of lipase I from Galactomyces geotrichum by codon optimisation in Pichia pastoris

Relatively poor heterologous protein yields have limited the commerical applications of Galactomyces geotrichum lipase I (GGl I) efficacy trials. To address this, we have redesigned the GGl I gene to preferentially match codon frequencies of Pichia pastoris (P. pastoris) while retaining the same amino acid sequence. The wild type and codon optimised GGl I (GGl I-wt and GGl I-op) were synthesised and cloned into pPICZαA with an N-terminal 6 × His tag sequence and expressed in P. pastoris X 33. The hydrolytic activity of GGl I-op was 150 U/mL, whereas the activity of the GGl I-wt could not be detected. GGl I-op recombinant proteins were purified by Ni-affinity chromatography and then characterised. The identity and purity of GGl I were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry and Western blot analysis. Enzymatic deglycosylation was used to show that the lipase is a glycosylated protein, containing ∼10% sugar. The molecular weight (MW) of the GGl I secreted by recombinant P. pastoris was approximated at 63 kDa. The optimum pH and temperature of the recombinant lipase were 8.0 and 35 °C, respectively. The enzyme was active over a broad pH range (7.0–9.0) and temperature range (20 °C–45 °C). The lipase showed high activity toward medium- and long-chain fatty acid methyl esters (C8-C16) and retained much of its activity in the presence of Tween-80 and Trition X-100. Lipase activity was stimulated by Mg2+, Ca2+, Mn2+ and Cu2+ and inhibited by Fe2+, Fe3+, Zn2+ and Co2+. This lipase may prove useful to the detergent industry and in organic synthesis reactions.

Accurate differentiation of Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS analysis.

The increasing number of infections caused by nontuberculous mycobacteria (NTM) has prompted the need for rapid and precise identification methods of these pathogens. Several studies report the applicability of MALDI-TOF mass spectrometry (MS) for identification of NTM. However, some closely related species have very similar spectral mass fingerprints, and until recently, Mycobacterium chimaera and M. intracellulare could not be separated from each other by MALDI-TOF MS. The conventional identification methods used in routine diagnostics have similar limitations. Recently, the differentiation of these two species within the Mycobacterium avium complex has become increasingly important due to reports of M. chimaera infections related to open heart surgery in Europe and in the USA. In this report, a method for the distinct differentiation of M. chimaera and M. intracellulare using a more detailed analysis of MALDI-TOF mass spectra is presented. Species-specific peaks could be identified and it was possible to assign all isolates (100 %) from reference strain collections as well as clinical isolates to the correct species. We have developed a model for the accurate identification of M. chimaera and M. intracellulare by MALDI-TOF MS. This approach has the potential for routine use in microbiology laboratories, as the model itself can be easily implemented into the software of the currently available systems by MALDI-TOF MS manufacturers.

A glycoproteomic approach reveals that the S-layer glycoprotein of Lactobacillus kefiri CIDCA 83111 is O- and N-glycosylated

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471–476, with a Glc5–8GalA2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species. SignificanceA detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp.

Abnormal glycosylation in Joubert syndrome type 10

BackgroundThe discovery of disease pathogenesis requires systematic agnostic screening of multiple homeostatic processes that may become deregulated. We illustrate this principle in the evaluation and diagnosis of a 5-year-old boy with Joubert syndrome type 10 (JBTS10). He carried the OFD1 mutation p.Gln886Lysfs*2 (NM_003611.2: c.2656del) and manifested features of Joubert syndrome.MethodsWe integrated exome sequencing, MALDI-TOF mass spectrometry analyses of plasma and cultured dermal fibroblasts glycomes, and full clinical evaluation of the proband. Analyses of cilia formation and lectin staining were performed by immunofluorescence. Measurement of cellular nucleotide sugar levels was performed with high-performance anion-exchange chromatography with pulsed amperometric detection. Statistical analyses utilized the Student’s and Fisher’s exact t tests.ResultsGlycome analyses of plasma and cultured dermal fibroblasts identified abnormal N- and O-linked glycosylation profiles. These findings replicated in two unrelated males with OFD1 mutations. Cultured fibroblasts from affected individuals had a defect in ciliogenesis. The proband’s fibroblasts also had an abnormally elevated nuclear sialylation signature and increased total cellular levels of CMP-sialic acid. Ciliogenesis and each glycosylation anomaly were rescued by expression of wild-type OFD1.ConclusionsThe rescue of ciliogenesis and glycosylation upon reintroduction of WT OFD1 suggests that both contribute to the pathogenesis of JBTS10.

Abortion and mortality in farm mink (Neovison vison) associated with feed-born Clostridium limosum

Disease in mink clinically characterized by abortion and increased mortality among pregnant female mink on 28 Danish farms was observed during April and May 2015. Most of these farms suffered extensive disease problems, including a significant increase in the number of mated females without litters. Pathological, microbiological and molecular biological methods were applied to investigate the cause of disease. Necropsies of animals found dead revealed fragile and partially dissolved (liquefying) uterine tissue, with the presence of Gram positive rod-shaped bacteria. These slow growing bacteria were isolated by anaerobic culturing and identified as Clostridium limosum by both MALDI-TOF mass spectrometry analysis and 16S rRNA gene sequencing. All the performed tests for relevant differential diagnoses were negative. Foodborne disease was indicated because all the affected farms were served by the same feed factory. A specific PCR-based analysis was developed for positive identification of C. limosum and used to screen archived feed samples from the implicated feed factory. Both C. limosum 16S rRNA genes and C. limosum collagenase genes were identified in both mixed feed and more specifically in raw chicken carcass used as one of the components in the mixed feed, which was therefore identified as the most likely source of contamination. Based on the results of this investigation it is concluded that C. limosum can be associated with abortion and increased mortality in pregnant mink females and it is consequently recommended that raw materials contaminated with C. limosum should be avoided in mink feed, in particular during the whelping season.

Proteomic based approach for characterizing 4-hydroxy-2-nonenal induced oxidation of buffalo (Bubalus bubalis) and goat (Capra hircus) meat myoglobins.

BackgroundMyoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins.MethodsPresent study characterize the Mb extracted from water buffalo and goat cardiac muscles using two-dimensional gel electrophoresis (2DE), OFFGEL electrophoresis and mass spectrometry (MS). Purified buffalo and goat bright red oxymyoglobin were reacted with HNE in-vitro at physiological pH (7.4) and temperature (37 °C) conditions and the formation of oxidised brown metmyoglobin was measured. The Mb-HNE adducts were detected using MALDI-TOF MS, whereas specific sites of adduction was determined using ESI-QTOF MS/MS.ResultsPurified buffalo and goat Mb samples revealed a molecular mass of 17,043.6 and 16,899.9 Daltons, respectively. The 2DE analysis exhibited 65 (sarcoplasmic protein extract) and 6 (pure Mb) differentially expressed (P < 0.05) protein spots between buffalo and goat samples. OFFGEL electrophoresis revealed an isoelectric point of 6.77 and 7.35 respectively, for buffalo and goat Mb’s. In-vitro incubation of HNE with bright red buffalo and goat oxymyoglobin’s at pH 7.4 and 37 °C resulted in pronounced (P < 0.05) oxidation and formation of brown metmyoglobin. MALDI-TOF MS analysis of Mb-HNE reaction mix revealed covalent binding (via Michael addition) of 3 and 5 molecules of HNE with buffalo and goat Oxy-Mb’s, respectively. ESI-QTOF MS/MS identified seven and nine histidine (HIS) residues of Mb that were readily adducted by HNE in buffalo and goat, respectively.ConclusionThe study demonstrated better redox stability of buffalo Mb than goat Mb. Our findings confirm the hypothesis that relative effect of HNE was greater for Mb’s with 12 ± 1 HIS residues than Mb’s with 9 HIS residues and helps meat processors in developing species-specific processing strategies to reduce the color variability.

MALDI-TOF mass spectrometry analysis of proteins and lipids in Escherichia coli exposed to copper ions and nanoparticles.

Escherichia coli (E. coli) is one of the most important foodborne pathogens to the food industry responsible for diseases as bloody diarrhea, hemorrhagic colitis and life-threatening hemolytic-uremic syndrome. For controlling and eliminating E. coli, metal nano-antimicrobials (NAMs) are frequently used as bioactive systems for applications in food treatments. Most NAMs provide controlled release of metal ions, eventually slowing down or completely inhibiting the growth of undesired microorganisms. Nonetheless, their antimicrobial action is not totally unraveled and is strongly dependent on metal properties and environmental conditions. In this work, we propose the use of matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry as a powerful tool for direct, time efficient, plausible identification of the cell membrane damage in bacterial strains exposed to copper-based antimicrobial agents, such as soluble salts (chosen as simplified AM material) and copper nanoparticles. E. coli ATCC 25922 strain was selected as 'training bacterium' to set up some critical experimental parameters (i.e. cell concentration, selection of the MALDI matrix, optimal solvent composition, sample preparation method) for the MS analyses. The resulting procedure was then used to attain both protein and lipid fingerprints from E. coli after exposure to different loadings of Cu salts and NPs. Interestingly, bacteria exposed to copper showed over-expression of copper binding proteins and degradation of lipids when treated with soluble salt. These findings were completed with other investigations, such as microbiological experiments. Copyright © 2016 John Wiley & Sons, Ltd.

Epigenetic changes in localized gastric cancer: the role of RUNX3 in tumor progression and the immune microenvironment.

Gastric cancer (GC) pathogenesis involves genetic, epigenetic and environmental factors. Epigenetic alterations, such as DNA methylation are considered pivotal in the inactivation of tumor-related genes. We assessed a methylation panel of 5 genes to study their association to GC progression and microsatellite instability (MSI), and studied the role of RUNX3 in GC pathogenesis and the tumor immune microenvironment.The methylation status of 47 promoter-CpG islands was studied through MALDI-TOF mass spectrometry analysis in 35 Microsatellite stable (MSS) GC, 26 MSI, and 18 cancer-free samples (CFS), and 6 MSS GC and 4 MSI GC cell lines. We also studied RUNX3 expression by immunohistochemistry (IHC) in 40 samples, and validated differences in methylation levels between tumor, normal, and immune tissue in 14 additional samples.Unsupervised hierarchical clustering of methylation levels revealed no distinct subgroups between MSI and MSS samples or cell lines. CFSs clustered together showing higher levels of RUNX3 methylation compared to GC samples. RUNX3 showed protein silencing in cancer and normal mucosa, compared to inflammatory peritumoural infiltrate in almost all cases, showing a non-lymphocytic predominant pattern and being correlated with epigenetic silencing.Our results show aberrant promoter's methylation in APC, CDH1, CDKN2A, MLH1 and RUNX3 associated with GC, as well as a non-lymphocytic predominant infiltrate with high expression of RUNX3. Deep study of RUNX3 inflammation signaling could help in understanding inflammation and immune activation in the tumor microenvironment.

Optimal turnaround time for direct identification of microorganisms by mass spectrometry in blood culture

IntroductionDuring the past few years, several studies describing direct identification of bacteria from blood culture using mass spectrometry have been published.These methods cannot, however, be easily integrated into a common laboratory workflow because of the high hands-on time they require. In this paper, we propose a new method of identification with a short hands-on time and a turnaround time shorter than 15min. Materials and methodsPositive blood bottles were homogenised and 600μL of blood were transferred to an Eppendorf tube where 600μL of lysis buffer were added. After homogenisation, a centrifugation step of 4min at 10,500g was performed and the supernatant was discarded. The pellet was then washed and loaded in quadruplicate into wells of a Vitek® MS-DS plate. Each well was covered with a saturated matrix solution and a MALDI-TOF mass spectrometry analysis was performed. Species were identified using the software Myla 3.2.0-2. ResultsWe analysed 266 positive blood culture bottles. A microorganism grew in 261 cultures, while five bottles remained sterile after 48h of incubation in subculture. Our method reaches a probability of detection at the species level of 77.8% (203/261) with a positive predictive value of 99.5% (202/203). ConclusionWe developed a new method for the identification of microorganisms using mass spectrometry, directly performed from a positive blood culture. This method has short hands-on time and turnaround time and can easily take place in the workflow of a laboratory, with comparable results in performance with other methods reported in the literature.

ACE-Inhibitory and Antioxidant Activities of Peptide Fragments Obtained from Tomato Processing By-Products Fermented Using Bacillus subtilis: Effect of Amino Acid Composition and Peptides Molecular Mass Distribution.

The effects of amino acid composition and peptide molecular mass on ACE-inhibitory and antioxidant activities of protein fragments obtained from tomato waste fermented using Bacillus subtilis were evaluated. The addition of B. subtilis increased the relative amounts of aromatic and positively-charged amino acids which have been described to influence the biological activities of peptide fragments. IC50 values of hydrolysates for ACE-inhibitory and 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities were found to be 1.5 and 8.2mg/mL, respectively. Size-exclusion chromatography (SEC) pattern of the hydrolysate indicated the breakdown of parent proteins to smaller peptides with molecular weights mainly below 1400Da. MALDI-TOF mass spectrometry analysis revealed that the highest ACE-inhibitory activity was due to peptides showing molecular mass range 500-800Da, while the most active antioxidant peptides were found to be mainly at the two different peptide weight ranges 500-800Da and 1200-1500Da.

Bacteriocinogenic Potential of Enterococcus faecium Isolated from Wine.

A total of 145 lactic acid bacteria isolated from a variety of Turkish red wines during malolactic fermentation were screened to find bacteriocin-producing strains. Among them, 14 isolates of Enterococcus faecium were identified to produce bacteriocins. PCR screening revealed that some isolates harbored entA and entB genes while some harbored entA, entB and entP genes. An isolate designated as Ent. faecium H46 was selected to characterize its bacteriocins. The bacteriocins were purified to homogeneity from culture supernatant by Amberlite XAD-16, cation-exchange and reverse-phase chromatography. MALDI-TOF mass spectrometry analysis identified the bacteriocins as enterocin A and enterocin B. The presence of Ent. faecium is noteworthy since it is not associated with wine fermentation. However, it has been reported as an important wine spoilage organism due to its potential to produce tyramine. Although species of Enterococcus is not known as wine bacteria, contamination by Ent. faecium may arise from grapes or wineries equipments used for wine production.

Simultaneous Determination of Multiple microRNA Levels Utilizing Biotinylated Dideoxynucleotides and Mass Spectrometry.

MicroRNAs (miRNAs) are important regulators of gene translation and have been suggested as potent biomarkers in various disease states. In this study, we established an efficient method for simultaneous determination of multiple miRNA levels, employing the previously developed SPC-SBE (solid phase capture-single base extension) approach and MALDI-TOF mass spectrometry (MS). In this approach, we first perform reverse transcription of miRNAs extracted using stem-loop primers. Then the cDNA is co-amplified with competitors, synthetic oligonucleotides whose sequences precisely match cDNA except for one base, and the amplicons serve as templates for a multiplexed SBE reaction. Extension products are isolated using SPC and quantitatively analyzed with MALDI-TOF MS to determine multiple miRNA levels. Here we demonstrated concurrent analysis of four miRNA levels utilizing the approach. Furthermore, we showed the presented method significantly facilitated MS analysis of peak area ratio owing to SPC. The SPC process allowed effective removal of irrelevant reaction components prior to MS and promoted MS sample purification. Data obtained in this study was verified with RT-qPCR and agreement was shown on one order of magnitude scale, suggesting the SPC-SBE and MS approach has strong potential as a viable tool for high throughput miRNA analysis.

Intact spore MALDI-TOF mass spectrometry and proteomic analysis of Puccinia pathogenic fungi

The aim of this work was to develop a method for the identification of pathogens causing rust diseases of crops using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells or spores (IC/IS). All optimizations were performed with Puccinia triticina, the causal agent of wheat leaf rust. Experiments included selection of washing solvents for spores, finding of an optimal concentration of spores in suspension and the most suitable matrix system as well as an evaluation of different sample preparation techniques. The best results were obtained when the spores were washed with acetonitrile/0.1% (v/v) trifluoroacetic acid, 7:3, v/v. A mixture of ferulic and sinapinic acids (5:15mgml−1) dissolved in acetonitrile/2.5% (v/v) trifluoroacetic acid, 7:3, v/v, was found optimal for the deposition of samples (50μg spores per μl) by two-layer volume technique. The optimized protocol was subsequently applied to other Puccinia species (Puccinia graminis, Puccinia striiformis and Puccinia coronata). Together with the use of the software BIOSPEAN, not only different species but also various pathotypes of the same species, which differ in their virulence, could be discriminated. There were 108 and 29 proteins identified from P. striiformis and P. graminis spores, respectively, after an acidic extraction in the matrix solvent mimicking the sample preparation for MALDI. Besides the presence of ribosomal proteins, histones, regulatory proteins and enzymes, also extracellular proteins participating in the pathogenesis were found. Finally, for both species, several proteins were assigned to signals in typical mass spectrometric profiles and suggested as diagnostic markers.

Detailed MALDI-TOF Mass spectrometry analysis for the discrimination of Candida haemulonii complex species

Introduction Candida haemulonii is now considered to be a complex of two species and one variety : C. haemulonii , Candida duobushaemulonii and the variety Candida haemulonii var. vulnera . The correct identification (ID) of these species is clinically relevant, since resistance to azole derivatives has been reported and amphotericin B has poor in vitro activity against C. duobushaemulonii isolates. Initial evaluation using MALDI-TOF MS for the ID of the C. haemulonii complex species provided promising results, but the discrimination of C. haemulonii from the variety vulnera was problematic, and the performance of the VITEK MS remains unevaluated. Objectives Evaluation of the MALDI-TOF MS performance for the ID of C. haemulonii species ID with two platforms and its’ databases and softwares. Methods 14 C. haemulonii sensu stricto , 9 C. haemulonii var. vulnera and 10 C. duobushaemulonii from hospitals belonging to the Medical school from the University of Sao Paulo were analyzed (ID by ITS1 sequence analysis). A set of reference strains from the CBS-KNAW collection was also included : C. haemulonii CBS5149 T, C. duobushaemulonii CBS7798 T, C. duobushaemulonii CBS7799, and for specificity control, the close related species Candida pseudohaemulonii (CBS10004 and CBS12370). The isolates/strains were cultured on Sabouraud plates and incubated for 48 h at 30 °C before MALDI-TOF MS analysis. Protein extraction protocol with ethanol and formic acid 70 % was carried out according to the Bruker's recommendations. Measurements were performed on a Microflex LT and its standard database (database 3.3.1), and VITEK MS instrument equipped with both IVD and RUO (SARAMIS) databases. To differentiate C. haemulonii (target group) from the variety vulnera (control group), mass spectra analysis models were created with the ClinProTools 3.0 software (Bruker). For each model, the recognition capability (RC) and cross validation (CV) percentage was generated. For single peak analysis, the AUC of each peak for the discrimination of target group from control group was directly obtained from the ClinProTools software. For the peaks with the highest AUC, the detection performances were checked using FlexAnalysis 3.4 (Bruker Daltonics). The signal-to-noise ratios (SN) of the peaks were exported to SPSS 18.0, ROC curves were constructed, and their optimal cut-off values were determined. Results All C. haemulonii sensu stricto isolates/strain had correct species assignment by both Bruker and VITEK MS IVD databases. For the species C. duobushaemulonii, the Bruker database gave correct species ID for 75 % of the isolates/strains. VITEK MS IVD analysis misidentified all C. duobushaemulonii and C. pseudohaemulonii isolates/strains as C. haemulonii (99 % of confidence level). The VITEK MS SARAMIS was unable to give genus/species ID for all isolates/strains. Discrimination of the isolates belonging to the C. haemulonii sensu stricto and to the variety vulnera was not possible with all systems and databases. The ClinProTools models showed values of CV and RC above ≥90 % for the discrimination of target group from control group. The most discriminative peaks were 5107, 6878 and 13,750 m/z (AUC >0,9), with sensibility and specificity of 88,6 %, 80,8 %, 85,8 %, and 96,6 %, 92,9 %, 93,8 %, respectively. The SN cut-off values from the peaks 5107, 6878 and 13,750 m/z for the discrimination of the target group from the control group were 2,5, 3,9 and 6,3, respectively.

Abstract 546: Characterization of the I4399M Variant of Apolipoprotein(a): Implications for Altered Prothrombotic Properties of Lipoprotein(a)

Elevated plasma levels of lipoprotein(a) (Lp(a)) are a causal risk factor for CHD. Lp(a) closely resembles LDL, but contains an additional glycoprotein apolipoprotein(a) (apo(a)) that is structurally homologous to the fibrinolytic proenzyme plasminogen. Key sequence differences render the protease-like domain in apo(a) catalytically inactive. This has led to speculation that Lp(a) can oppose the fibrinolytic functions of plasminogen. A single nucleotide polymorphism (SNP) in the LPA gene encoding apo(a) results in an Ile to Met substitution at position 4399 in the protease-like domain. In population studies, this variant has been correlated with elevated plasma Lp(a) levels and with higher CHD risk, an effect that has been suggested to arise from effects of the variant on fibrin clot properties. We undertook a functional characterization of the effect of the I4399M substitution in apo(a). Molecular dynamics simulations of wild-type (wt) apo(a) and the Met variant revealed a shift from a buried (Ile) to slightly exposed (Met) environment, allowing for potential modification of the Met. Indeed, MALDI-TOF mass spectrometry analysis demonstrated the presence of a methionine sulfoxide moiety at this position in the Met variant. When 17-kringle recombinant forms of apo(a) were included in a plasma clot lysis assay, both the wt apo(a) and Met variant inhibited lysis, but the Met variant had a 50% greater effect. However, the Met variant was equally as efficient as wt apo(a) in inhibiting plasminogen activation on a fibrin surface. Apo(a) was also able to significantly shorten coagulation time for clots made from either purified fibrin or lipoprotein-deficient plasma, with the Met variant having twice as large an effect as wt apo(a). Morever, the Met variant resulted in greater turbidity of the clots whereas wt apo(a) had no effect. In agreement with these findings, SEM and confocal microscopy of fibrin clots showed that compared to wt apo(a), the I4399M variant resulted in significant alteration of the fibrin network, with a 4-fold increase in fibrin fiber width. Together, our data suggest that the Met4399 variant differs structurally from wt apo(a), which may underlie key differences related to its effects on fibrin clot architecture and fibrinolysis.

Highly Stereocontrolled Ring-Opening Polymerization of Racemic Alkyl β-Malolactonates Mediated by Yttrium [Amino-alkoxy-bis(phenolate)] Complexes.

Yttrium [amino-alkoxy-bis(phenolate)]amido complexes have been used for the ring-opening polymerization (ROP) of racemic alkyl β-malolactonates (4-alkoxycarbonyl-2-oxetanones, rac-MLA(R) s) bearing an allyl (All), benzyl (Bz) or methyl (Me) lateral ester function. The nature of the ortho-substituent on the phenolate rings in the metal ancillary dictated the stereocontrol of the ROP, and consequently the syndiotactic enrichment of the resulting polyesters. ROP promoted by catalysts with halogen (Cl, Br)-disubstituted ligands allowed the first reported synthesis of highly syndiotactic PMLA(R) s (Pr ≥ 0.95); conversely, catalysts bearing bulky alkyl and aryl ortho-substituted ligands proved largely ineffective. All polymers have been characterized by (1) H and (13) C{(1) H} NMR spectroscopy, MALDI-ToF mass spectrometry and DSC analyses. Statistical and thermal analyses enabled the rationalization of the chain-end control mechanism. Whereas the stereocontrol of the polymerization obeyed a Markov first-order (Mk1) model for the ROP of rac-MLA(Bz) and rac-MLA(All) , the ROP of rac-MLA(Me) led to a chain end-control of Markov second-order type (Mk2). DFT computations suggest that the high stereocontrol ability featured by catalysts bearing Cl- and Br-substituted ligands does not likely originate from halogen bonding between the halogen substituent and the growing polyester chain.

Extensive cultivation of soil and water samples yields various pathogens in patients with cystic fibrosis but not Burkholderia multivorans

BackgroundWhile the epidemiology of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients suggests that Burkholderia multivorans is acquired from environmental sources, this species has rarely been isolated from soil and water samples. MethodsMultiple isolation strategies were applied to water and soil samples that were previously shown to be B. multivorans PCR positive. These included direct plating and liquid enrichment procedures and the use of selective media, acclimatizing recovery and co-cultivation with CF sputum. MALDI-TOF mass spectrometry and sequence analysis of 16S rRNA and housekeeping genes were used to identify all isolates. ResultsNone of the approaches yielded B. multivorans isolates. Other Burkholderia species, several Gram-negative non-fermenting bacteria (including Cupriavidus, Inquilinus, Pandoraea, Pseudomonas and Stenotrophomonas) and rapidly growing mycobacteria (including Mycobacterium chelonae) were all isolated from water and soil samples. ConclusionsThe use of Bcc isolation media yielded a surprisingly wide array of rare but often clinically relevant CF pathogens, confirming that soil and water are reservoirs of these infectious agents.

Structural studies of Helix aspersa agglutinin complexed with GalNAc: A lectin that serves as a diagnostic tool

Lectins belong to a differentiated group of proteins known to possess sugar-binding properties. Due to this fact, they are interesting research targets in medical diagnostics. Helix aspersa agglutinin (HAA) is a lectin that recognizes the epitopes containing α-d-N-acetylgalactosamine (GalNAc), which is present at the surface of metastatic cancer cells. Although several reports have already described the use of HAA as a diagnostic tool, this protein was not characterized on the molecular level. Here, we present for the first time the structural information about lectin isolated from mucus of Helix aspersa (garden snail). The amino acid sequence of this agglutinin was determined by Edman degradation and tertiary as well as quaternary structure by X-ray crystallography. The high resolution crystal structure (1.38Å) and MALDI-TOF mass spectrometry analysis provide the detailed information about a large part of the HAA natural glycan chain. The topology of the GalNAc binding cleft and interaction with lectin are very well defined in the structure and fully confirmed by STD HSQC NMR spectroscopy. Together, this provides structural clues regarding HAA specificity and opens possibilities to rational modifications of this important diagnostic tool.