High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis of base-specific cleavage products is an efficient, highly accurate tool for the detection of single base sequence variations. We describe the first application of this comparative sequencing strategy for automated high-throughput mutation detection in microbial genomes. The method was applied to identify DNA sequence changes that occurred in Escherichia coli K-12 MG1655 during laboratory adaptive evolution to new optimal growth phenotypes. Experiments were based on a genome-scale in silico model of E. coli metabolism and growth. This model computes several phenotypic functions and predicts optimal growth rates. To identify mutations underlying a 40-d adaptive laboratory evolution on glycerol, we resequenced 4.4% of the E. coli-K12 MG1655 genome in several clones picked at the end of the evolutionary process. The 1.54-Mb screen was completed in 13.5 h. This resequencing study is the largest reported by MALDI-TOF mass spectrometry to date. Ten mutations in 40 clones and three deviations from the reference sequence were detected. Mutations were predominantly found within the glycerol kinase gene. Functional characterization of the most prominent mutation shows its metabolic impact on the process of adaptive evolution. All sequence changes were independently confirmed by genotyping and Sanger-sequencing. We demonstrate that comparative sequencing by base-specific cleavage and MALDI-TOF mass spectrometry is an automated, fast, and highly accurate alternative to capillary sequencing.     

Carbapenemase activity detection by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.     

Comparison of rifaximin and lactulose for the treatment of hepatic encephalopathy: a prospective randomized study

Rifaximin has been reported to be effective for the treatment of hepatic encephalopathy (HE) in Europe. However, it is unknown whether Rifaximin is effective for the treatment of HE in Koreans, therefore we conducted a open-label prospective randomized study to evaluate the efficacy of rifaximin versus lactulose in Korean patients. Fifty-four patients with liver cirrhosis and hepatic encephalopathy were enrolled. Thirty-two patients were randomized to receive rifaximin and 22 to receive lactulose both over a 7-day periods. Before and at the end of treatment, gradation of blood ammonia, flapping tremor, mental status, number connection test (NCT) were performed and estimation of HE indexes determined. Both rifaximin and lactulose were effective in the majority of patients (84.4% and 95.4%, respectively, p = 0.315). Blood NH3, flapping tremor, mental status, and NCT was significantly improved by rifaximin and lactulose, and the post- treatment levels of these measures were similar for the rifaximin and lactulose-treated groups, as was the HE index (rifaximin group (10.0 --> 4.2, p = 0.000); lactulose group (11.3 --> 5.0, p = 0.000)). One patient treated with rifaximin complained of abdominal pain, which was easily controlled. There was no episode of renal function impairment in either treatment group. Rifaximin proved to be as safe and as effective as lactulose for the treatment of Korean patients with hepatic encephalopathy.     

Efficacy of levofloxacin and rifaximin based quadruple therapy in Helicobacter pylori associated gastroduodenal disease: a double-blind, randomized controlled trial

The aim of this study was to evaluate the efficacy of levofloxacin and rifaximin based quadruple regimen as first-line treatment for Helicobacter pylori infection. A prospectively randomized, double-blinded, parallel group, comparative study was performed. Three hundred consecutive H. pylori positive patients were randomized to receive: omeprazole, amoxicillin, clarithromycin (OAC); omeprazole, amoxicillin, levofloxacin (OAL); and omeprazole, amoxicillin, levofloxacin, rifaximin (OAL-R). The eradication rates in the intention to treat (ITT) and per protocol (PP) analyses were: OAC, 77.8% and 85.6%; OAL, 65.3% and 73.6%; and OAL-R, 74.5% and 80.2%. The eradication rate achieved with OAC was higher than with OAL on the ITT (P = 0.05) and PP analysis (P = 0.04). OAL-R regimen was not inferior to OAC. The frequency of moderate to severe adverse effects was significantly higher in OAC treatment group. Especially, diarrhea was most common complaint, and there was a significantly low rate of moderate to severe diarrhea with the rifaximin containing regimen. In conclusion, the levofloxacin and rifaximin based regimen comes up to the standard triple therapy, but has a limited efficacy in a Korean cohort. The rifaximin containing regimen has a very high safety profile for H. pylori eradication therapy.     

Influence of rifaximin treatment on the susceptibility of intestinal Gram-negative flora and enterococci

The development of rifaximin- and rifampicin-resistant intestinal coliforms was studied in 27 subjects receiving rifaximin for 3 days by plating stool samples on media containing rifaximin 200 mg/L or rifampicin 64 mg/L before treatment (day 0), after treatment was completed (day 3), and after a further 2 days (day 5). The susceptibility of enterococci grown on day 0 and day 3 was also studied in 71 subjects. Significant increases in antimicrobial-resistant coliform flora were not seen in either the rifaximin-treated or the placebo-treated subjects. Enterococci recovered pre- and post-treatment showed similar susceptibilities. Rifaximin did not select for significant resistance in the Gram-negative and Gram-positive intestinal flora during therapy.     

激光显微切割联合质谱分析继发淀粉样变性患者肾组织SAA蛋白

目的利用激光显微切割联合质谱(LMD/MS)技术分析强直性脊柱炎(AS)合并继发性淀粉样变患者肾活检组织标本血清淀粉样物质A(SAA)蛋白亚型及氨基酸突变序列。方法甲醛固定肾活检组织,脱蜡后行刚果红染色,选取刚果红染色阳性区域进行质谱分析,通过数据分析软件对质谱结果进行整合评估,并将患者SAA蛋白氨基酸序列与变异蛋白数据库氨基酸序列进行比对确定是否有变异蛋白。结果质谱鉴定到高丰度的SAA1及SAA2蛋白,同时有血清淀粉样蛋白P及载脂蛋白E,数据库比对未检测到SAA1及SAA2蛋白的变异序列。结论本研究首次鉴定到了AS合并淀粉样变肾组织中的SAA1及SAA2蛋白,丰富了AS淀粉样变的发病机制,为将来AA型淀粉样变性的精准分型提供新的方法。     

Characterization of the major membrane protein of virulent Mycobacterium tuberculosis.

A protein with a molecular mass of 19 kDa was isolated and purified from enriched membrane fractions of the virulent Erdman strain of Mycobacterium tuberculosis. The protein is different from another 19-kDa protein, a lipoprotein, that was recently described (D. B. Young and T. R. Garbe, Res. Microbiol. 142:55-65, 1991). The sequencing strategy applied to this major membrane protein employed four different endoproteinases and resulted in sufficient overlapping peptide sequences for assignment of the entire protein sequence. Electron spray ionization mass spectrometry demonstrated a measured mass of 16,100, deviating from the predicted mass by only 2.86 atomic mass units. The sequence of this protein is unique. However, some similarities with other low-molecular-weight heat shock proteins were observed. Immunoblotting indicated that this protein is highly expressed in the virulent strains of M. tuberculosis. Its application to sera from patients with pulmonary tuberculosis showed promise as a serodiagnostic tool.     

Carbapenem-hydrolyzing oxacillinase in high resistant strains of Acinetobacter baumannii isolated from India

Acinetobacter baumannii, a Gram negative bacterium causes nosocomial infections including bacteremia, secondary meningitis and urinary tract infections. Increased resistance of A. baumannii has been global concern. Till recently, carbapenems, latest generation of β-lactams are used for treating infections caused by A. baumannii. Emerging resistance to carbapenem class is an immediate threat to mankind. The objective of present study is to understand the growing carbapenem resistance of A. baumannii. By using iso-electric focusing followed by (in-gel) nitrocefin assay of carbapenem resistant strains of A. baumannii, we could identify three β-lactamases with pIs in the range 5.4-9.5. Expression of the β-lactamase with a pI ≈ 8.5, was found only in very high carbapenem resistant (MIC for imipenem 128 μg/ml) strains. On PCR analysis and sequencing of PCR product, this β-lactamase was confirmed to be OXA-51. Identification of this protein from IEF gel was reconfirmed with the help of Liquid chromatography and Tandem mass spectrometry (LC-MS/MS). Based on the amino acid sequence, OXA-51 found to be a 30 kDa β-lactamase containing conserved functional motifs of class D serine β-lactamase. In the present study, we have established the emergence of OXA-51 in clinical strains of A. baumannii in India which suggests its role in carbapenem resistance.     

Purification and HPLC-MS analysis of a naturally processed HCMV-derived peptide isolated from the HEK-293T/HLA-E+/Ul40+ cell transfectants and presented at the cell surface in the context of HLA-E

A new method for isolation and characterization of peptides presented in the context of the nonclassical human leukocytes antigen (HLA) class I molecule HLA-E was developed. A combination of different chromatographic steps coupled with electrospray mass spectrometry allowed us to detect the presence of small amounts of a naturally processed human Cytomegalovirus (HCMV)-derived peptide isolated from the HEK-293T/HLA-E+/UL40+ transfected cells of from HELA cell line. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). This approach provides a versatile and sensitive method for direct identification of MHC class I-binding peptides that might be derive from different pathogen or tumor-associated proteins.     

Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.

The occurrence of glycosylated proteins in Mycobacterium tuberculosis has been widely reported. However, unequivocal proof for the presence of true glycosylated amino acids within these proteins has not been demonstrated, and such evidence is essential because of the predominance of soluble lipoglycans and glycolipids in all mycobacterial extracts. We have confirmed the presence of several putative glycoproteins in subcellular fractions of M. tuberculosis by reaction with the lectin concanavalin A. One such product, with a molecular mass of 45 kDa, was purified from the culture filtrate. Compositional analysis demonstrated that the protein was rich in proline and that mannose, galactose, glucose, and arabinose together represented about 4% of the total mass. The 45-kDa glycoprotein was subjected to proteolytic digestion with either the Asp-N or the Glu-C endopeptidase or subtilisin, peptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and glycopeptides were identified by reaction with concanavalin A. Peptides were further separated, and when they were analyzed by liquid chromatography-electrospray mass spectrometry for neutral losses of hexoses (162 mass units), four peptides were identified, indicating that these were glycosylated with hexose residues. One peptide, with an average molecular mass of 1,516 atomic mass units (AMU), exhibited a loss of two hexose units. The N-terminal sequence of the 1,516-AMU glycopeptide was determined to be DPEPAPPVP, which was identical to the sequence of the amino terminus of the mature protein, DPEPAP PVPXTA. Furthermore, analysis of the glycopeptide by secondary ion mass spectrometry demonstrated that the complete sequence of the glycopeptide was DPEPAPPVPTTA. From this, it was determined that the 10th amino acid, threonine, was O-glycosidically linked to a disaccharide composed of two hexose residues, probably mannose. This report establishes that true, O-glycosylated proteins exist in mycobacteria.     

Novel modification of lipid A of Francisella tularensis

We have investigated the lipid A of Francisella tularensis subsp. holarctica strain 1547-57, a type B strain, by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, nanoelectrospray quadrupole ion-trap mass spectrometry, and chemical methods. In accordance with the previously published structures of the lipid A from F. tularensis live vaccine strain (LVS) (ATCC 29684) (E. Vinogradov et al., Eur. J. Biochem. 269:6112-6118, 2002), all of the major lipid A forms from strain 1547-57 were tetraacylated. As in the LVS strain, the major fatty acids detected in the F. tularensis 1547-57 lipid A sample included 3-hydroxyoctadecanoic acid, 3-hydroxyhexadecanoic acid, hexadecanoic acid, and tetradecanoic acid. However, several of the lipid A components present in strain 1547-57 were of higher molecular weight than the previously published structures. A major component with an M(r) of 1,666 was found to contain three C(18:0)(3-OH) fatty acids, one C(16:0) fatty acid, one phosphate group, and one 161-Da moiety. This 161-Da moiety could be removed from the lipid A by treatment with aqueous hydrofluoric acid and was identified as galactosamine following peracetylation and analysis by gas chromatography-mass spectrometry. Detailed investigations of the M(r)-1,666 species by ion-trap mass spectrometry with multiple stages of fragmentation suggested that the galactosamine-1-phosphate was linked to the reducing terminus of the lipid A. Similar to the modification of lipid A with arabinosamine, lipopolysaccharide species from F. tularensis containing a phosphate-linked galactosamine could potentially influence its intracellular survival by conferring resistance to antimicrobial peptides.     

Waist circumference predicts metabolic cardiovascular risk in postmenopausal Chinese women

OBJECTIVE: The purpose of the study is to compare the differences in metabolic cardiovascular risk factors among postmenopausal Chinese women with or without abdominal obesity. DESIGN: The study is a cross-sectional, population-based, comparative cohort study. Each participant received anthropometric measurements and a 75-g oral glucose tolerance test after an overnight fast. The homeostasis model assessment for insulin resistance and the insulin sensitivity index, ISI, were used as measures of insulin resistance. A "metabolic cardiovascular risk score" was calculated from fasting insulin, glucose, lipids, and blood pressure. General linear models (GLM) were fit to examine the relation of waist circumference (WC) to insulin resistance and metabolic risk scores.(0,120). RESULTS: According to the International Obesity Task Force obesity criteria for Asians, 57 women had abdominal obesity (WC >/= 80 cm), and 58 had WCs less than 80 cm. The two groups were comparable in demographic variables and body mass index (BMI). The women with larger WCs were more insulin-resistant than their counterparts. The metabolic risk scores were significantly higher in women with abdominal obesity than in those without it. The results from the GLM showed that WC was an independent predictor of insulin resistance and metabolic risk scores after controlling for demographic variables (0.06- and 0.29-unit increases in homeostasis model assessment for insulin resistance and metabolic risk scores per 1 cm change in WC). Moreover, BMI neither correlated with metabolic risk scores nor affected the WC effects on insulin resistance and metabolic risk scores in the GLM. CONCLUSIONS: Postmenopausal Chinese women with abdominal obesity may carry higher metabolic cardiovascular risk than those without it. It is WC, not BMI, that predicts metabolic cardiovascular risk factors in these women.     

Human catalytic RNA- and DNA-hydrolyzing antibodies

Sequencing of anti-vancomycin monoclonal antibody (mAb) Fab region (48,000 Da) was carried out using liquid chromatography–electrospray ionization ion trap mass spectrometry (LC/ESI-MS). Comprehensive strategies were employed to ensure complete sequence coverage. The sequence information was obtained from the spectra of collision-induced dissociation (CID) (MS/MS) of the protonated proteolytic peptides resulting from multiple enzymatic digestions of reduced/S-carboxymethylated (RCM) light chain and Fd fragment. Database searching of the spectra against the published immunoglobulin G (IgG) sequences allowed the identification of all the peptides in constant domains as well as partial sequences in variable domains. The rest of the sequences were deduced by manual interpretation of the peptide tandem mass spectrometry (MS/MS) spectra. The analysis showed that the N-terminus of the heavy chain was modified by the conversion of a glutamine residue to pyroglutamic acid.     

4819例遗传代谢病检测结果分析

目的 为了解遗传代谢病的发病率,以便推动遗传代谢病的全面筛查,应用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)检测血氨基酸和酰基肉碱,联合气相色谱-串联质谱(gas chromatography-tandem mass spectrometry,GC-MS)技术检测尿液中有机酸,对氨基酸、有机酸代谢病及脂肪酸β氧化障碍进行筛查和诊断.方法 收集4819例(包括1388例新生儿及3431例疑似遗传代谢病高危儿童)血氨基酸和酰基肉碱检测结果及尿有机酸检测结果,分别利用LC-MS/MS检测了4778例干滤纸片和GC-MS检测了3004例尿标本.结果 通过遗传代谢病筛查共确诊88例(占所检测样本的1.83%,这88例均行LC-MS/MS和GC-MS检测),其中氨基酸代谢病9种,37例;有机酸代谢病7种,40例;脂肪酸β氧化障碍5种,11例.结论 联合LC-MS/MS及GC-MS能快速对遗传代谢病进行筛查和诊断.     

Metabolomic analysis reveals hepatic metabolite perturbations in citrin/mitochondrial glycerol-3-phosphate dehydrogenase double-knockout mice, a model of human citrin deficiency

The citrin/mitochondrial glycerol-3-phosphate dehydrogenase (mGPD) double-knockout mouse displays phenotypic attributes of both neonatal intrahepatic cholestasis and adult-onset type II citrullinemia, making it a suitable model of human citrin deficiency. In the present study, we investigated metabolic disturbances in the livers of wild-type, citrin (Ctrn) knockout, mGPD knockout, and Ctrn/mGPD double-knockout mice following oral sucrose versus saline administration using metabolomic approaches. By using gas chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry, we found three general groupings of metabolite changes in the livers of the double-knockout mice following sucrose administration that were subsequently confirmed using liquid chromatography/mass spectrometry or enzymatic methods: a marked increase of hepatic glycerol 3-phosphate, a generalized decrease of hepatic tricarboxylic acid cycle intermediates, and alterations of hepatic amino acid levels related to the urea cycle or lysine catabolism including marked increases in citrulline and lysine. Furthermore, concurrent oral administration of sodium pyruvate with sucrose ameliorated the hyperammonemia induced by sucrose, as had been shown previously, as well as almost completely normalizing the hepatic metabolite perturbations found. Overall, we have identified additional metabolic disturbances in double-KO mice following oral sucrose administration, and provided further evidence for the therapeutic use of sodium pyruvate in our mouse model of citrin deficiency.     

Determination of Rifaximin Treatment Period According to Lactulose Breath Test Values in Nonconstipated Irritable Bowel Syndrome Subjects

Small intestinal bacterial overgrowth (SIBO) can partly explain irritable bowel syndrome (IBS), and rifaximin has been observed to improve abdominal symptoms in nonconstipated IBS patients. However, there are few reports on the association of the rifaximin treatment periods with the results of a lactulose breath test (LBT). Therefore, we performed a retrospective review of patient charts to investigate the relation between the rifaximin treatment periods with LBT results in nonconstipated IBS patients. We also evaluated the time to achieve a symptomatic improvement in the IBS patients as compared to the changes in the LBT. We reviewed the charts for patients who showed IBS symptoms with documented positive results for LBT during their initial visit and who had a follow-up LBT after treatment with rifaximin. The LBT values were compared to the subjects'' symptom scores. A total of 102 subjects had a follow-up LBT to assess LBT normalization. The subjects were divided into groups according to treatment periods of 4 weeks (n = 36), 8 weeks (n = 43), and 12 weeks (n = 23). The groups with a longer treatment exhibited an increase in the hydrogen gas value at 90 min and its sum during 90 min at the initial LBT. There were significant differences in hydrogen gas value at 90 min and in its sum during 90 min at the initial LBT between the groups treated for 4 and 12 weeks. The most significant treatment response was observed during the first 4 weeks for all treatment groups. Symptomatic improvement occurred earlier than LBT normalization in the treatment period over 4 weeks. The results indicate that different rifaximin treatment periods are needed in accordance with LBT levels to effectively eradicate SIBO.

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Mass spectrometry of peptides

Recent progress in biochemistry has shown the occurrence of many important peptides, among antibiotics, immunostimulants, hormones and neuromediators. The mass spectrometric study of these components by classical ionization techniques such as electron impact, chemical ionization or field desorption require a prior chemical derivatization because of their amphoteric properties, their low volatility and low thermostability. Recent improvements in methods of ionization, fast atom bombardment, secondary ion mass spectrometry, or mass spectrometry/mass spectrometry have made it possible to study the structure of peptides with more than 15 amino acid residues and eased mass spectra interpretation. Through the different methodologies described in this review three characteristic informations can be obtained concerning the molecular mass of peptides, the nature of constituent amino acids and the peptidic sequence and even quantitative measurements can be performed.     

Metabolic oligosaccharide engineering of Plasmodium falciparum intraerythrocytic stages allows direct glycolipid analysis by mass spectrometry

A recent addition to the arsenal of tools for glycome analysis is the use of metabolic labels that allow covalent tagging of glycans with imaging probes. In this work we show that N-azidoglucosamine was successfully incorporated into glycolipidic structures of Plasmodium falciparum intraerythrocytic stages. The ability to tag glycoconjugates selectively with a fluorescent reporter group permits TLC detection of the glycolipids providing a new method to quantify dynamic changes in the glycosylation pattern and facilitating direct mass spectrometry analyses. Presence of glycosylphosphatidylinositol and glycosphingolipid structures was determined in the different extracts. Furthermore, the fluorescent tag was used as internal matrix for the MALDI experiment making even easier the analysis.     

Tranylcypromine ('Parnate') overdose: measurement of tranylcypromine concentrations and MAO inhibitory activity and identification of amphetamines in plasma.

A case of tranylcypromine overdose is reported. Tranylcypromine, amphetamine methamphetamine and phenylethylamine were detected in the plasma by gas chromatography and their identity confirmed by mass spectrometry. The data suggested that the amphetamines were metabolic products of tranylcypromine. Platelet monoamine oxidase activity was more than 95% inhibited during the 72 h after the overdose despite complete clinical recovery by that time. The possible role of amphetamines and phenylethylamine in causing the clinical manifestations of tranylcypromine overdose is discussed.