OBJECTIVETo investigate the mutations of SLC22A5 gene in patients with systemic primary carnitine deficiency (CDSP).

METHODSHigh liquid chromatography tandem mass spectrometry (HPLC/MS/MS) was applied to screen congenital genetic metabolic disease and eight patients with CDSP were diagnosed among 77 511 samples. The SLC22A5 gene mutation was detected using massarray technology and sanger sequencing. Using SIFT and PolyPhen-2 to predict the function of protein for novel variations.

RESULTSTotal detection rate of gene mutation is 100% in the eight patients with CDSP. Seven patients had compound heterozygous mutations and one patient had homozygous mutations. Six different mutations were identified, including one nonsense mutation [c.760C>T(p.R254X)] and five missense mutations[c.51C>G(p.F17L), c.250T>A(p.Y84N), c.1195C>T(p.R399W), c.1196G>A(p.R399Q), c.1400C>G(p.S467C)]. The c.250T>A(p.Y84N) was a novel variation, the novel variation was predicted to have affected protein structure and function. The c.760C>T (p.R254X)was the most frequently seen mutation, which was followed by the c.1400C>G(p.S467C).

CONCLUSIONThis study confirmed the diagnosis of eight patients with CDSP on the gene level. Six mutations were found in the SLC22A5 gene, including one novel mutation which expanded the mutational spectrum of the SLC22A5 gene.

Adult ; Amino Acid Sequence ; Base Sequence ; Cardiomyopathies ; diagnosis ; genetics ; metabolism ; Carnitine ; deficiency ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; Female ; Gene Frequency ; Genotype ; Humans ; Hyperammonemia ; diagnosis ; genetics ; metabolism ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis ; genetics ; metabolism ; Mutation ; Organic Cation Transport Proteins ; genetics ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Homology, Amino Acid ; Solute Carrier Family 22 Member 5 ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and accurate identification of clinical yeast isolates. In-tube formic acid/acetonitrile (FA/ACN) extraction is recommended prior to the analysis with MALDI Biotyper, but the direct on-plate FA extraction is simpler. We compared the Biotyper with the VITEK MS for the identification of various clinically relevant yeast species, focusing on the use of the FA extraction method. METHODS: We analyzed 309 clinical isolates of 42 yeast species (four common Candida species, Cryptococcus neoformans, and 37 uncommon yeast species) using the Biotyper and VITEK MS systems. FA extraction was used initially for all isolates. If ‘no identification' result was obtained following the initial FA extraction, these samples were then retested by using FA (both systems, additive FA) or FA/ACN (Biotyper only, additive FA/ACN) extraction. These results were compared with those obtained by sequence-based identification. RESULTS: Both systems correctly identified all 158 isolates of the four common Candida species after the initial FA extraction. The Biotyper correctly identified 8.7%, 30.4%, and 100% of 23 C. neoformans isolates after performing initial FA, additive FA, and FA/ACN extractions, respectively, while VITEK MS identified all C. neoformans isolates after the initial FA extraction. Both systems had comparable identification rates of 37 uncommon yeast species (128 isolates), following the initial FA (Biotyper, 74.2%; VITEK MS, 73.4%) or additive FA (Biotyper, 82.0%; VITEK MS, 73.4%). CONCLUSIONS: The identification rate of most common and uncommon yeast isolates is comparable between simple FA extraction/Biotyper method and VITEK MS methods, but FA/ACN extraction is necessary for C. neoformans identification by Biotyper.
Candida ; Cryptococcus neoformans ; Mass Spectrometry* ; Methods* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Yeasts*

The platelet-rich plasma (PRP) and antimicrobial peptides neutrophil extract (AMP extract) were prepared from rabbit neutrophils as two autologous blood-derived preparations, which could be applied locally to enhance healing process of tissues. Both preparations were analyzed using the MALDI TOF method for accurate qualitative assay. Growth factors (PDGF and VEGF) and microbicidal protein were reported in PRP. In AMP extract a-defensins, namely; NP-1, -2, -3a, -3b, -4, and -5 and cathelicidins represented among other by 15-kDa antibacterial protein (p15s) were detected. In the second part of our study the influence of antimicrobial extract on macrophages in vitro was tested. Then, degranulation of neutrophils in vitro and generation of reactive intermediates by these cells under the influence of AMP extract were assessed. As estimated, the addition of AMP extract into cultures of macrophages decreased superoxide anion generation after 5 days of incubation. Furthermore, AMP extract inhibited degranulation and respiratory burst in neutrophils, therefore in this regard it suppress proinflammatory effect of two studied populations of leukocytes.
Cathelicidins ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Leukocytes ; Macrophages ; Methods ; Neutrophils* ; Peptides ; Platelet-Rich Plasma* ; Respiratory Burst ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Superoxides

OBJECTIVES: To analyse the genetic polymorphisms of 66 biallelic genetic markers on Y chromosome in Eastern Chinese Han population, and evaluate their values in forensic application. METHODS: Genotyping of 66 biallelic genetic markers on Y chromosome was studied in 205 unrelated males of Eastern Chinese Han population by multiplex PCR combined matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS）. The allele frequencies on the loci to be tested were calculated by direct counting method, and the gene diversity （GD） and haplotype diversity （HD） were calculated by corresponding formulas. The haplotypes of this system were tested by software Arlequin v3.5.2.2 and the comparison of population genetics were analyzed. RESULTS: A total of 60 biallelic genetic markers on Y chromosome were polymorphic in males of Eastern Chinese Han population, and the ranges of GD were from 0.038 5 to 0.501 9. Eighty-five different haplotypes were observed and the HD was 0.970 3. The differences of partial SNP loci between the Han population of Eastern China and that of Xinjiang and Guangdong were statistically significance. CONCLUSIONS Sixty biallelic genetic markers and the detection system can complementally provide genetic information in kinship testing and individual identification. The MALDI-TOF-MS technology is able to type biallelic genetic markers.
Asian People/genetics* ; China ; Chromosomes, Human, Y/genetics* ; Gene Frequency ; Genetic Markers/genetics* ; Genetic Variation ; Genetics, Population ; Genotype ; Haplotypes/genetics* ; Humans ; Male ; Multiplex Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide/genetics* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVE: To establish a diagnostic model for diffuse axonal injury (DAI) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To screen the proteins or peptides associated with DAI for providing the biomarkers with theoretic foundation. METHODS: Fifteen male Sprague-Dawley rats were randomly divided into DAI group (n = 10) and control group (n = 5). The protein or peptide expression profiles of rat brain stem were detected by MALDI-TOF-MS. ClinProTools 2.2 software was used to find specific peaks, and a diagnostic model was established by the genetic algorithm. RESULTS: There were significant differences in 61 peaks of DAI group (P < 0.05), 9 peaks were down-regulated and 52 up-regulated. The diagnostic model was established based on 5 different peaks. The specificity and sensitivity of cross validation was 96.14% and 95.98%; while the specificity and sensitivity of blind validation showed was 73.33% and 70.00%, respectively. CONCLUSION A specific and sensitive diagnostic model of DAI can be established by MALDI-TOF-MS to provide a potential value for determining DAI in forensic practice.
Animals ; Biomarkers ; Brain Stem/metabolism* ; Diffuse Axonal Injury/diagnosis* ; Down-Regulation ; Male ; Peptides/blood* ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Sensitivity and Specificity ; Software ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods* ; Up-Regulation

Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS) has been a classical technique for studying proteomics in present and a tool for analyzing the distribution of proteins and small molecules within biological tissue sections. MALDI-TOF-IMS can analyze multiple unknown compounds in biological tissue sections simultaneously through a single measurement which can obtain molecule imaging of the tissue while maintaining the integrity of cellular and molecules in tissue. In recent years, imaging mass spectrometry technique develops relatively quickly in all biomedical domain. This paper based on the relevant data and reviews the present developing level of MALDI-TOF-IMS, the principle of imaging mass spectrometry, methology and the prospect in forensic pathology.
Diagnostic Imaging ; Forensic Sciences/methods* ; Humans ; Male ; Proteins ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUND: By conventional methods, the identification of anaerobic bacteria is more time consuming and requires more expertise than the identification of aerobic bacteria. Although the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) systems are relatively less studied, they have been reported to be a promising method for the identification of anaerobes. We evaluated the performance of the VITEK MS in vitro diagnostic (IVD; 1.1 database; bioMerieux, France) in the identification of anaerobes. METHODS: We used 274 anaerobic bacteria isolated from various clinical specimens. The results for the identification of the bacteria by VITEK MS were compared to those obtained by phenotypic methods and 16S rRNA gene sequencing. RESULTS: Among the 249 isolates included in the IVD database, the VITEK MS correctly identified 209 (83.9%) isolates to the species level and an additional 18 (7.2%) at the genus level. In particular, the VITEK MS correctly identified clinically relevant and frequently isolated anaerobic bacteria to the species level. The remaining 22 isolates (8.8%) were either not identified or misidentified. The VITEK MS could not identify the 25 isolates absent from the IVD database to the species level. CONCLUSIONS: The VITEK MS showed reliable identifications for clinically relevant anaerobic bacteria.
Bacteria, Anaerobic/*genetics/isolation & purification ; Bacterial Typing Techniques/*instrumentation/*methods ; Body Fluids/microbiology ; Databases, Genetic ; Humans ; RNA, Ribosomal, 16S/*analysis/metabolism ; Sequence Analysis, DNA ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

BACKGROUND: Acinetobacter species are the leading cause of bloodstream infection (BSI), but their correct identification is challenging. We evaluated the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based VITEK MS (bioMerieux, France), and two automated systems, VITEK 2 (bioMerieux) and MicroScan (Siemens, USA) for identification of Acinetobacter BSI isolates. METHODS: A total of 187 BSI isolates recovered at a university hospital in Korea between 2010 and 2012 were analyzed. The identification results obtained using VITEK MS and two automated systems were compared with those of rpoB sequencing. RESULTS: Of 187 isolates analyzed, 176 were identified to the species level by rpoB sequencing: the Acinetobacter baumannii group (ABG; 101 A. baumannii, 43 A. nosocomialis, 10 A. pittii isolates) was most commonly identified (82.4%), followed by Acinetobacter genomic species 13BJ/14TU (5.3%), A. ursingii (2.1%), A. soli (2.1%), A. bereziniae (1.1%), and A. junii (1.1%). Correct identification rates to the species group (ABG) level or the species level was comparable among the three systems (VITEK MS, 90.3%; VITEK 2, 89.2%; MicroScan, 86.9%). However, VITEK MS generated fewer misidentifications (0.6%) than VITEK 2 (10.8%) and MicroScan (13.1%) (P<0.001). In addition, VITEK MS demonstrated higher specificity (100%) for discrimination between ABG and non-ABG isolates than the other systems (both, 31.8%) (P<0.001). CONCLUSIONS: The VITEK MS system is superior to the VITEK 2 and MicroScan systems for identification of Acinetobacter BSI isolates, with fewer misidentifications and better discrimination between the ABG and non-ABG isolates.
Acinetobacter/*genetics/isolation & purification ; Acinetobacter Infections/diagnosis/microbiology ; Bacterial Proteins/genetics ; Bacterial Typing Techniques/*instrumentation/*methods ; Blood/*microbiology ; DNA, Bacterial/*analysis/metabolism ; Databases, Genetic ; Humans ; *Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

PURPOSE: Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. MATERIALS AND METHODS: The cells were treated with 300 microM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. RESULTS: Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (alpha1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (epsilon subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (epsilon subunit) are unknown in relation to carcinogenesis. CONCLUSION: Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.
Animals ; Electrophoresis, Gel, Two-Dimensional/*methods ; Fibroblasts/*metabolism/pathology ; HSP70 Heat-Shock Proteins ; Humans ; Mice ; NIH 3T3 Cells ; Neoplasms/*metabolism/pathology ; Nitric Oxide Donors ; Nitroso Compounds ; Proteins/analysis/*metabolism ; Proteomics/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

PURPOSE: The genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB). MATERIALS AND METHODS: We analyzed 65 Aeromonas isolates recovered from patients at a university hospital in Korea between 1996 and 2012. The isolates were recovered from frozen states and tested using the following four methods: a conventional biochemical method, 16S rRNA sequencing, housekeeping gene sequencing with phylogenetic analysis, and MALDI-TOF MS. RESULTS: The conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level. CONCLUSION: The most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.
Aeromonas/classification/*genetics/isolation & purification ; DNA, Bacterial/genetics ; Genes, Essential/*genetics ; Humans ; Molecular Typing/*methods ; Phylogeny ; RNA, Ribosomal, 16S/*genetics ; Republic of Korea ; Sensitivity and Specificity ; Sequence Analysis, DNA/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods