Odontogenic tumors are rare lesions with unknown etiopathogenesis. Most of them are benign, but local aggressiveness, infiltrative potential, and high recurrence rate characterize some entities. The MAP-kinase pathway activation can represent a primary critical event in odontogenic tumorigenesis. Especially, the BRAF V600E mutation has been involved in 80-90% of ameloblastic lesions, offering a biological rationale for developing new targeted therapies. The study aims to evaluate the BRAF V600E mutation in odontogenic lesions, comparing three different detection methods and focusing on the Sequenom MassARRAY System. 81 surgical samples of odontogenic lesions were subjected to immunohistochemical analysis, Sanger Sequencing, and Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (Sequenom). The BRAF V600E mutation was revealed only in ameloblastoma samples. Moreover, the presence of BRAF V600E was significantly associated with the mandibular site (ρ = 0.627; P value <0.001) and the unicystic histotype (ρ = 0.299, P value <0.001). However, any significant difference of 10-years disease-free survival time was not revealed. Finally, Sequenom showed to be a 100% sensitive and 98.1% specific, suggesting its high-performance diagnostic accuracy. These results suggest the MAP-kinase pathway could contribute to ameloblastic tumorigenesis. Moreover, they could indicate the anatomical specificity of the driving mutations of mandibular ameloblastomas, providing a biological rational for developing new targeted therapies. Finally, the high diagnostic accuracy of Sequenom was confirmed.
Ameloblastoma/pathology* ; Carcinogenesis ; Humans ; Mitogen-Activated Protein Kinases/genetics* ; Mutation ; Odontogenic Tumors/pathology* ; Proto-Oncogene Proteins B-raf/metabolism* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

In this study, a method was established for in-situ visualization of metabolite distribution in the rhizome of Paris polyphylla var. yunnanensis. To be specific, through matrix-assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI), the spatial locations of steroidal saponins, amino acids, organic acids, phytosterols, phytoecdysones, nucleosides, and esters in rhizome of the medicinal plant were directly analyzed, and six unknown compounds with differential distribution in rhizome tissues were identified. The specific procedure is as follows: preparation of rhizome tissue section, matrix screening and optimization, and MALDI-MSI analysis. The results showed that the steroidal saponins were mainly distributed in the central, amino acids in epidermis and cortex, low-molecular-weight organic acids in central epidermis, phytosterols in the epidermis and lateral cortex, the phytoecdysones in epidermis and cortex, nucleosides(uneven distribution) in epidermis and cortex, growth hormones around the epidermis and cortex, particularly outside the cortex, and esters in cortex with unobvious difference among different tissues. In this study, the spatial distribution of meta-bolites in the rhizome of P. polyphylla var. yunnanensis was characterized for the first time. The result can serve as a reference for identifying and extracting endogenous metabolites of P. polyphylla var. yunnanensis, exploring the synthesis and metabolism mechanisms of the metabolites, and evaluating the quality of medicinal materials.
Liliaceae/chemistry* ; Melanthiaceae ; Rhizome/chemistry* ; Saponins/analysis* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Aims: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used to identify Candida spp. in diagnostic laboratories due to its strength in providing accurate information results, speed and cost-effectiveness. However, its accuracy varies on instrument platform, reference database, sample preparation techniques and interlaboratory comparisons. Therefore, the use of MALDI-TOF MS for species identification was evaluated against traditional biochemical identification, namely BrillianceTM Candida and Remel RapIDTM Yeast Plus System. Methodology and results: To evaluate and compare identification efficiency, turnaround time and consumable cost, 194 clinical isolates of Candida were collected. The results showed overall 85.6% concordant identification between two methods with 94.9-99.5% and 100% accuracy in traditional and MALDI-TOF MS, respectively, in the identification of four common Candida species; C. albicans, C. tropicalis, C. parapsilosis sensu stricto and C. glabrata sensu stricto. Other Candida species were also identified with 85.6% and 97.5% accuracy rates by traditional and MALDI-TOF MS, respectively. Additionally, identification using MALDI-TOF MS reduced overall turnaround time and cost by approximately 99.8% and 86.5%, respectively. Conclusion, significance and impact of study This study highlights the performance of MALDI-TOF MS, which is more accurate in identifying Candida spp. with a less hands-on approach, cheaper cost and shorter turnaround time.
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Candida ; Tertiary Care Centers

Mass spectrometry imaging （MSI） is a new imaging technology that can simultaneously detect and record the spatial distribution information of multiple molecules on the sample surface without labeling. The main principle of MSI is to combine mass spectrometry with imaging technology and irradiate the sample slice with ion beam or laser to ionize the molecules on its surface, obtain the mass spectrometry signal through the detector, convert the obtained data into pixel points by the imaging software, and then construct the spatial distribution image of the target compound on the tissue surface. The sample preparation for MSI include： sample collection and storage, tissue section, tissue pretreatment, selection and application of matrix. At present, this technology has been widely used in the fields of biomedicine, new drug development and proteomics, and its application in the field of forensic toxicology has also gradually attracted attention. This article reviews the principles and sample preparation process of MSI, describes the application of MSI in abused substances and metabolites of various material matrices, herbal mixtures, latent fingerprints, hair and animal and plant tissues, and discusses the prospects of the application of this technology in forensic toxicology, in order to provide ideas and references for the application of MSI technology in forensic toxicology.
Animals ; Diagnostic Imaging ; Forensic Toxicology ; Humans ; Mass Spectrometry ; Plants ; Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) is a novel technique for in-situ distribution of various substances in tissue without labeling. This technique is increasingly applied to the study of medicinal plants owing to its high spatial resolution and its potential of in-situ analysis in small molecules. In this study, the structural information and their fragmentation patterns of the midazole alkaloids(1,3-dibenzyl-4,5-dimethylimidazolium chloride and 1,3-dibenzyl-2,4,5-trimethylimi-dazolium chloride) and benzylglucosinolate in the medicinal plant Maca(Lepdium meyeni) root were analyzed by ultra-high-performance liquid phase combined with LTQ-Orbitrap mass spectrometry(UHPLC-HR-MS). The localization of these active ingredients in the cross-sections of Maca root was performed by MALDI-MSI. These results demonstrated that the two types of imidazole alkaloids had a similar distributed pattern. They were located more in the cortex and the periderm than those in the medulla of a lateral root, while the localization of benzylglucosinolate was concentrated in the center of the root rather than in the cortex and the periderm. The precise spatial distribution of various secondary metabolites in tissue provides an important scientific basis for the accumulation of medicinal plant active ingredients in tissues. In addition, this imaging method is a promising technique for the rapid evaluation and identification of the active ingredients of traditional Chinese medicine in plant tissues, as well as assisting the research on the processing of medicinal plants.
Chromatography, High Pressure Liquid ; Lepidium/chemistry* ; Phytochemicals/analysis* ; Plant Roots/chemistry* ; Plants, Medicinal/chemistry* ; Secondary Metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

To establish a two-dimensional gel electrophoresis (2-DE) map for comparative proteomic analysis of rat spinal cord with chronic morphine tolerance, and to detect differentially expression proteins that are associated with chronic morphine tolerance.
 Methods: Sixteen male SD rats received the intrathecal catheterization operation and they were randomly divided into a morphine tolerance group (MT group, n=8) and a saline group (NS group, n=8). The lumbar enlargement segments of the MT group and the NS group spinal cord were harvested and proteins were separated by 2-DE. Differential proteome profiles were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The 2-DE maps were visualized after coomassie blue staining and analyzed using PDQuest analysis software. Identification of differential protein spots was conducted by MALDI-TOF-MS, and the Mascot query software was used to search Swiss-Prot database for bioinformatics analysis. Western blotting was used to verify the expression of some differentially expressed proteins.
 Results: A total of 1 000 spots were identified in 2-DE maps of rat spinal cord tissues from the MT group and the NS group, and 36 proteins were significantly differentially expressed in the MT group compared with the NS group. Identification was conducted by MALDI-TOF-MS and Swiss-Prot database through Mascot query software, and a total of 14 proteins were obtained. Among them, 2 protein spots were down-regulated in the MT group compared with that in the NS group, and 12 protein spots were up-regulated in the MT group compared with that in the NS group. Two kinds of proteins (NUDAA, ENOG) were verified by Western blotting and the results were consistent with proteomics data.
 Conclusion: The optimized 2-DE profiles for the proteome of spinal cord tissue in rats with chronic morphine tolerance is established preliminarily, which showed that morphine tolerance can cause changes in the expression of various proteins in the spinal cord.
Animals ; Electrophoresis, Gel, Two-Dimensional ; Male ; Morphine ; Proteome ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Spinal Cord

BACKGROUND: This study was conducted to evaluate the impact of the media type used for direct identification of colonies on the surveillance culture of carbapenem-resistant Enterobacteriaceae (CRE) by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). METHODS: CRE surveillance culture isolates were subjected to species identification using the MALDI Biotyper (Bruker Daltonics, Germany) for 2 months starting in March 2017. Four types of media were evaluated: blood agar (BA), Mueller Hinton agar (MH), MacConkey agar (Mac), and MacConkey agar containing imipenem of 1 µg/mL (IMP-Mac). CRE-like colonies on IMP-Mac and their subculture colonies on the other media were tested after overnight incubation and extended incubation for one additional day. The percent identification and score value were analyzed for each media types and incubation time when the identification was correct at the genus level. RESULTS: A total of 117 isolates were identified as 84 Klebsiella pneumoniae, 12 Escherichia coli, 9 Enterobacter cloacae, 5 Klebsiella oxytoca, 4 Enterobacter aerogenes, and 2 Raoultella ornithinolytica. The successful identification rates (SIR) for BA and MH were 98.3% and 97.4% (P=0.9), respectively, while those for Mac and IMP-Mac were 82.1% (P < 0.001) and 70.9% (P < 0.001), respectively. After extended incubation, SIRs were decreased to 96.6%, 96.6% (P=1.0), 61.5% (P < 0.001), and 58.1% (P < 0.001) on BA, MH, Mac, and IMP-Mac, respectively. The average score values were significantly lower for Mac (2.017±0.22) and IMP-Mac (1.978±0.24) than for BA (2.213±0.16) (P < 0.001). CONCLUSIONS: The low performance of the MALDI Biotyper applied directly to the colonies grown on Mac or IMP-Mac indicates that subculture on BA or MH is preferable before identification by MALDI-TOF MS.
Agar ; Enterobacter aerogenes ; Enterobacter cloacae ; Enterobacteriaceae* ; Escherichia coli ; Imipenem ; Klebsiella oxytoca ; Klebsiella pneumoniae ; Mass Spectrometry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*

BACKGROUND: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. METHODS: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. RESULTS: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. CONCLUSIONS: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.
Actinomyces ; Agar ; Bacteria ; Bifidobacterium ; Bronchoalveolar Lavage Fluid* ; Bronchoalveolar Lavage* ; Bronchoscopy ; Clostridium ; Enterobacteriaceae ; Fungi ; Humans ; Lactobacillus ; Lung ; Mass Spectrometry ; Microbiota ; Neisseria ; Prevotella ; Respiratory System ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Staphylococcus ; Streptococcus ; Veillonella

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*