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1.
BMC Infect Dis ; 20(1): 687, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948147

RESUMO

BACKGROUND: Vogesella species are common aquatic, Gram-negative rod-shaped bacteria, originally described in 1997. Vogesella perlucida was first isolated from spring water in 2008. Furthermore, bacterial pathogenicity of Vogesella perlucida has never been reported. Here, we report the first case of rare Vogesella perlucida-induced bacteremia in an advanced-age patient with many basic diseases and history of dexamethasone abuse. CASE PRESENTATION: A 71-year-old female was admitted with inflamed upper and lower limbs, rubefaction, pain and fever (about 40 °C). She had been injured in a fall at a vegetable market and then touched river snails with her injury hands. A few days later, soft tissue infection of the patient developed and worsened. Non-pigmented colonies were isolated from blood cultures of the patient. Initially, Vogesella perlucida was wrongly identified as Sphingomonas paucimobilis by Vitek-2 system with GN card. Besides, we failed to obtain an acceptable identification by the MALDI-TOF analysis. Finally, the isolated strain was identified as Vogesella perlucida by 16S rRNA gene sequences. In addition, the patient recovered well after a continuous treatment of levofloxacin for 12 days. CONCLUSION: Traditional microbiological testing system may be inadequate in the diagnosis of rare pathogenic bacteria. Applications of molecular diagnostics techniques have great advantages in clinical microbiology laboratory. By using 16S rRNA gene sequence analysis, we report the the first case of rare Vogesella perlucida-induced bacteremia.


Assuntos
Bacteriemia/microbiologia , Betaproteobacteria/patogenicidade , Infecções dos Tecidos Moles/microbiologia , Idoso , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Técnicas de Tipagem Bacteriana , Betaproteobacteria/classificação , Betaproteobacteria/genética , Betaproteobacteria/isolamento & purificação , Feminino , Humanos , Levofloxacino/uso terapêutico , RNA Ribossômico 16S/genética , Infecções dos Tecidos Moles/tratamento farmacológico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vancomicina/uso terapêutico
2.
J Am Soc Mass Spectrom ; 31(10): 2013-2024, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-32880453

RESUMO

As corona virus disease 2019 (COVID-19) is a rapidly growing public health crisis across the world, our knowledge of meaningful diagnostic tests and treatment for severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) is still evolving. This novel coronavirus disease COVID-19 can be diagnosed using RT-PCR, but inadequate access to reagents, equipment, and a nonspecific target has slowed disease detection and management. Precision medicine, individualized patient care, requires suitable diagnostics approaches to tackle the challenging aspects of viral outbreaks where many tests are needed in a rapid and deployable approach. Mass spectrometry (MS)-based technologies such as proteomics, glycomics, lipidomics, and metabolomics have been applied in disease outbreaks for identification of infectious disease agents such as virus and bacteria and the molecular phenomena associated with pathogenesis. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) is widely used in clinical diagnostics in the United States and Europe for bacterial pathogen identification. Paper spray ionization mass spectrometry (PSI-MS), a rapid ambient MS technique, has recently open a new opportunity for future clinical investigation to diagnose pathogens. Ultra-high-pressure liquid chromatography coupled high-resolution mass spectrometry (UHPLC-HRMS)-based metabolomics and lipidomics have been employed in large-scale biomedical research to discriminate infectious pathogens and uncover biomarkers associated with pathogenesis. PCR-MS has emerged as a new technology with the capability to directly identify known pathogens from the clinical specimens and the potential to identify genetic evidence of undiscovered pathogens. Moreover, miniaturized MS offers possible applications with relatively fast, highly sensitive, and potentially portable ways to analyze for viral compounds. However, beneficial aspects of these rapidly growing MS technologies in pandemics like COVID-19 outbreaks has been limited. Hence, this perspective gives a brief of the existing knowledge, current challenges, and opportunities for MS-based techniques as a promising avenue in studying emerging pathogen outbreaks such as COVID-19.


Assuntos
Infecções por Coronavirus/etiologia , Lipidômica/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Pneumonia Viral/etiologia , Proteômica/métodos , Cromatografia Líquida de Alta Pressão , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Glicômica/métodos , Humanos , Pandemias , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
3.
PLoS One ; 15(8): e0234098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817616

RESUMO

In French Guiana, the malaria, a parasitic infection transmitted by Anopheline mosquitoes, remains a disease of public health importance. To prevent malaria transmission, the main effective way remains Anopheles control. For an effective control, accurate Anopheles species identification is indispensable to distinguish malaria vectors from non-vectors. Although, morphological and molecular methods are largely used, an innovative tool, based on protein pattern comparisons, the Matrix Assisted Laser Desorption / Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) profiling, emerged this last decade for arthropod identification. However, the limited mosquito fauna diversity of reference MS spectra remains one of the main drawback for its large usage. The aim of the present study was then to create and to share reference MS spectra for the identification of French Guiana Anopheline species. A total of eight distinct Anopheles species, among which four are malaria vectors, were collected in 6 areas. To improve Anopheles identification, two body parts, legs and thoraxes, were independently submitted to MS for the creation of respective reference MS spectra database (DB). This study underlined that double checking by MS enhanced the Anopheles identification confidence and rate of reliable classification. The sharing of this reference MS spectra DB should make easier Anopheles species monitoring in endemic malaria area to help malaria vector control or elimination programs.


Assuntos
Anopheles/classificação , Mosquitos Vetores/classificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anopheles/química , Guiana Francesa , Malária/classificação , Malária/transmissão , Especificidade da Espécie , Tórax
4.
Rev Chilena Infectol ; 37(3): 252-256, 2020 Jun.
Artigo em Espanhol | MEDLINE | ID: mdl-32853316

RESUMO

BACKGROUND: Mycobacterial diseases are very important both clinically and epidemiologically. Mycobacterium tuberculosis complex (MTBc) infections confer higher morbidity and mortality rate than non-tuberculous mycobacteria (NTM) infections. Traditional species identification techniques are based on phenotypic characteristics which take a long time by laborious processes and in occasions are no conclusive. Currently, most used techniques are based on molecular methods, which are accurate but are expensive and complex. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) is a simple, cheap and fast identification method based on comparing protein spectra with a reference database. AIM: To assess the performance of MALDI-TOF MS in the identification of MTBc and NTM, compared with molecular methods. METHODS: For that purpose, 28 isolates of 9 different species were analyzed through MALDI-TOF MS. RESULTS: 78.5% (22/28) of isolates were correctly identified, 100% (9/9) of rapidly growers NTM, 60% (9/15) of slow growing NTM and 100% (4/4) of MTBc. Every unidentified isolate (6/6) corresponded to M. avium/intracellulare complex. CONCLUSION: MALDI-TOF MS is fast, simple and cheaper than molecular methods and also has adequate accuracy.


Assuntos
Mycobacterium , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose
5.
BMC Infect Dis ; 20(1): 578, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32758181

RESUMO

BACKGROUND: Gram-positive anaerobic (GPA) bacteria inhabit different parts of the human body as commensals but can also cause bacteremia. In this retrospective observational study, we analyzed GPA bacteremia pathogens before (2013-2015) and after (2016-2018) the introduction of the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). METHOD: We conducted a retrospective observational study by searching the microbiology database to identify all positive GPA blood cultures of patients with GPA bacteremia diagnosed using the new technique, MALDI-TOF MS, between January 1, 2016 and December 31, 2018; and using a conventional phenotypic method between January 1, 2013 and December 31, 2015 at a single tertiary center in Japan. Parvimonas micra (P. micra) (17.5%) was the second most frequently identified GPA (MALDI-TOF MS); we then retrospectively reviewed electronic medical records for 25 P. micra bacteremia cases at our hospital. We also conducted a literature review of published cases in PubMed from January 1, 1980, until December 31, 2019; 27 cases were retrieved. RESULTS: Most cases of P. micra bacteremia were identified after 2015, both, at our institute and from the literature review. They were of mostly elderly patients and had comorbid conditions (malignancies and diabetes). In our cases, laryngeal pharynx (7/25, 28%) and gastrointestinal tract (GIT; 6/25, 24%) were identified as the most likely sources of bacteremia; however, the infection source was not identified in 9 cases (36%). P. micra bacteremia were frequently associated with spondylodiscitis (29.6%), oropharyngeal infection (25.9%), intra-abdominal abscess (14.8%), infective endocarditis (11.1%), septic pulmonary emboli (11.1%), and GIT infection (11.1%) in the literature review. Almost all cases were treated successfully with antibiotics and by abscess drainage. The 30-day mortalities were 4 and 3.7% for our cases and the literature cases, respectively. CONCLUSIONS: Infection sites of P. micra are predominantly associated with GIT, oropharyngeal, vertebral spine, intra-abdominal region, pulmonary, and heart valves. Patients with P. micra bacteremia could have good prognosis following appropriate treatment.


Assuntos
Bacteriemia/diagnóstico , Firmicutes/química , Bactérias Gram-Positivas/química , Infecções por Bactérias Gram-Positivas/sangue , Infecções por Bactérias Gram-Positivas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Hemocultura , Discite/microbiologia , Feminino , Firmicutes/isolamento & purificação , Trato Gastrointestinal/microbiologia , Bactérias Gram-Positivas/isolamento & purificação , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Japão , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Orofaringe/microbiologia , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Resultado do Tratamento , Adulto Jovem
6.
Nat Biotechnol ; 38(10): 1168-1173, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32733106

RESUMO

Detection of SARS-CoV-2 using RT-PCR and other advanced methods can achieve high accuracy. However, their application is limited in countries that lack sufficient resources to handle large-scale testing during the COVID-19 pandemic. Here, we describe a method to detect SARS-CoV-2 in nasal swabs using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and machine learning analysis. This approach uses equipment and expertise commonly found in clinical laboratories in developing countries. We obtained mass spectra from a total of 362 samples (211 SARS-CoV-2-positive and 151 negative by RT-PCR) without prior sample preparation from three different laboratories. We tested two feature selection methods and six machine learning approaches to identify the top performing analysis approaches and determine the accuracy of SARS-CoV-2 detection. The support vector machine model provided the highest accuracy (93.9%), with 7% false positives and 5% false negatives. Our results suggest that MALDI-MS and machine learning analysis can be used to reliably detect SARS-CoV-2 in nasal swab samples.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Algoritmos , Biotecnologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Países em Desenvolvimento , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Aprendizado de Máquina , Mucosa Nasal/virologia , Pandemias , Pneumonia Viral/epidemiologia , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/estatística & dados numéricos , Máquina de Vetores de Suporte
7.
Nat Commun ; 11(1): 4142, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32811831

RESUMO

Glycans are involved in various life processes and represent critical targets of biomedical developments. Nevertheless, the accessibility to long glycans with precise structures remains challenging. Here we report on the synthesis of glycans consisting of [→4)-α-Rha-(1 → 3)-ß-Man-(1 → ] repeating unit, which are relevant to the O-antigen of Bacteroides vulgatus, a common component of gut microbiota. The optimal combination of assembly strategy, protecting group arrangement, and glycosylation reaction has enabled us to synthesize up to a 128-mer glycan. The synthetic glycans are accurately characterized by advanced NMR and MS approaches, the 3D structures are defined, and their potent binding activity with human DC-SIGN, a receptor associated with the gut lymphoid tissue, is disclosed.


Assuntos
Bacteroides/química , Antígenos O/química , Polissacarídeos/síntese química , Bacteroides/imunologia , Bacteroides/metabolismo , Microbioma Gastrointestinal/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Espectroscopia de Ressonância Magnética , Antígenos O/imunologia , Antígenos O/metabolismo , Polissacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
BMC Infect Dis ; 20(1): 619, 2020 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-32831055

RESUMO

BACKGROUND: Neisseria macacae was discovered in the oral cavity of monkeys in 1983. In humans, it has been isolated from the upper respiratory tract of neutropenic patients. However, only two cases of N. macacae bacteremia have been reported in a 65-year-old man with infective endocarditis and a 5-month-old child with fever and petechiae. There are no reports of infections in cancer patients. Here, we present two cases of N. macacae bacteremia in cancer patients. CASE PRESENTATION: In the first case, a 42-year-old woman who underwent ovarian cancer surgery presented with duodenal invasion associated with multiple lymph node metastasis. N. macacae was isolated from her blood culture and identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). In the second case, a 69-year-old woman with a long-standing history of esophagogastric junction cancer presented with fever. She had stage IVB cancer with lung, bone, and multiple lymph node metastasis. The last chemotherapy was administered 5 weeks before N. macacae was detected using MALDI-TOF MS and nitrate test negative. In both cases, transthoracic echography showed no vegetation. Antibiotics were administered for 14 and 13 days in the first and second cases, respectively. In both cases, fever alleviated on day 4 of antibiotic administration. Both patients were discharged after their conditions improved. CONCLUSIONS: This, to our knowledge, is the first report of N. macacae bacteremia in cancer patients. Both patients, mucosal damage was observed in the upper gastrointestinal tract. Therefore, exclusion diagnosis suggested that bacteremia invasion was caused by mucosal rupture in both cases. Both cases responded well to treatment with ß-lactam antibiotics and improved after 2 weeks. Modifying the treatment based on the source of the infection may shorten the treatment period. Therefore, further research on N. macacae bacteremia is necessary. Immunocompromised patients such as those with cancer are susceptible to mucosal damage by unusual bacterial species such as N. macacae despite not having contact with monkeys.


Assuntos
Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Neisseria/patogenicidade , Adulto , Idoso , Antibacterianos/uso terapêutico , Hemocultura/métodos , Endocardite Bacteriana/microbiologia , Neoplasias Esofágicas/microbiologia , Junção Esofagogástrica/patologia , Feminino , Humanos , Masculino , Neisseria/genética , Neisseria/isolamento & purificação , Neoplasias Ovarianas/microbiologia , RNA Ribossômico 16S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
9.
J Chromatogr A ; 1626: 461335, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797820

RESUMO

On-spot fixed-charge derivatization has been suggested for the modification of α-amino acids for their analysis by thin layer chromatography/matrix-assisted laser desorption ionization (TLC/MALDI) mass spectrometry. The approach was based on post-chromatographic treatment of separated analytes by tris(2,6-dimethoxyphenyl)methenium salt and triethylamine. The reaction proceeded smoothly in mild conditions and gave rise to pink-red colored derivatives, containing permanent positive charge. Their MALDI mass spectra, recorded directly from TLC plates, revealed intense peaks corresponding to decarboxylated cationic parts. All derivatives are characterized by high ionization efficiency, which indicates the high sensitivity of the developed method for analyzing amino acids. Applicability of the method to analysis of amino acids was demonstrated on artificial mixtures and dietary supplement.


Assuntos
Aminoácidos/análise , Cromatografia em Camada Delgada/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Aminoácidos/química , Etilaminas/química
10.
J Chromatogr A ; 1627: 461422, 2020 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-32823117

RESUMO

Sialylation, an important form of glycosylation, is involved in many biological processes and plays an important role in the development of diseases. However, due to the low abundance among various glycosylation and lack of efficient enrichment method with high specificity, the study of sialylation remains a challenge. Herein, multi-histidine modified microspheres (MHM) were synthesized to enrich sialylated glycopeptides. It was found that MHM could selectively enrich sialylated glycopeptides from over 100 times of non-sialylated glycopeptides, which indicated MHM possessed good enrichment specificity towards sialylated glycopeptides. Furthermore, MHM were utilized to the large-scale analysis of protein sialylation, and 510 intact glycopeptides were identified with over 94.5% sialylated glycopeptide specificity from 4 µL human serum. The good specificity could be attributed to the synergistic effect by the electrostatic interaction and hydrophilic interaction. Hence, MHM could provide an alternative approach for the analysis of site-specific sialylation at proteome level from complex biological samples.


Assuntos
Glicopeptídeos/análise , Histidina/química , Ácido N-Acetilneuramínico/química , Sequência de Aminoácidos , Avidina/química , Fetuínas/química , Glicopeptídeos/sangue , Glicosilação , Humanos , Microesferas , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
J Med Microbiol ; 69(8): 1089-1094, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32692646

RESUMO

Introduction. The bla CTX-M-3 gene has rarely been reported in Morganella morganii strains and its genetic environment has not yet been investigated.Aim. To identify the bla CTX-M-3 gene in M. morganii isolated from swine and characterize its genetic environment.Methodology. A M. morganii isolate (named MM1L5) from a deceased swine was identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and subjected to antimicrobial susceptibility testing. The bla genes were detected and then the genetic location and environment of bla CTX-M-3 were investigated by Southern blot and PCR mapping, respectively. The M. morganii bla CTX-M-3 gene was cloned and expressed in Escherichia coli.Results. Isolate MM1L5 harboured the bla CTX-M-3 and bla TEM-1 genes. The bla CTX-M-3 gene, located on the chromosome, was co-carried with an IS26 and bla TEM-1 gene by a novel 6361 bp IS26-flanked composite transposon, designated Tn6741. This transposon consisted of a novel bla CTX-M-3-containing module, IS26-ΔISEcp1-bla CTX-M-3-Δorf477-IS26 (named Tn6710), and a bla TEM-1-containing module, IS26-Δorf477-bla TEM-1-tnpR-IS26, differing from previous reports. Phylogenetic analysis showed a significant variation based on the sequence of Tn6741, as compared to those of other related transposons. Interestingly, although the cloned bla CTX-M-3 gene could confer resistance to ceftiofur, cefquinome, ceftriaxone and cefotaxime, one amino acid substitution (Ile-142-Thr) resulted in a significant reduction of resistance to these antimicrobials.Conclusion. This is the first time that bla CTX-M-3 has been identified on a chromosome from a M. morganii isolate. Furthermore, the bla CTX-M-3 gene was located with an IS26 element and bla TEM-1 gene on a novel IS26-flanked composite transposon, Tn6741, suggesting that Tn6741 might act as a reservoir for the bla CTX-M-3 and bla TEM-1 genes and may become an important vehicle for their dissemination among M. morganii.


Assuntos
Elementos de DNA Transponíveis/genética , Morganella morganii/genética , beta-Lactamases/genética , Animais , Anti-Infecciosos/farmacologia , Sequência de Bases , Clonagem Molecular , Morganella morganii/classificação , Morganella morganii/efeitos dos fármacos , Filogenia , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos
12.
Nat Commun ; 11(1): 3556, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32678093

RESUMO

Early cancer detection greatly increases the chances for successful treatment, but available diagnostics for some tumours, including lung adenocarcinoma (LA), are limited. An ideal early-stage diagnosis of LA for large-scale clinical use must address quick detection, low invasiveness, and high performance. Here, we conduct machine learning of serum metabolic patterns to detect early-stage LA. We extract direct metabolic patterns by the optimized ferric particle-assisted laser desorption/ionization mass spectrometry within 1 s using only 50 nL of serum. We define a metabolic range of 100-400 Da with 143 m/z features. We diagnose early-stage LA with sensitivity~70-90% and specificity~90-93% through the sparse regression machine learning of patterns. We identify a biomarker panel of seven metabolites and relevant pathways to distinguish early-stage LA from controls (p < 0.05). Our approach advances the design of metabolic analysis for early cancer detection and holds promise as an efficient test for low-cost rollout to clinics.


Assuntos
Adenocarcinoma de Pulmão/sangue , Biomarcadores Tumorais/sangue , Neoplasias Pulmonares/sangue , Aprendizado de Máquina , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Detecção Precoce de Câncer , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metabolômica , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
13.
Prostate ; 80(13): 1071-1086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32687633

RESUMO

BACKGROUND: The emergence of reactive stroma is a hallmark of prostate cancer (PCa) progression and a potential source for prognostic and diagnostic markers of PCa. Collagen is a main component of reactive stroma and changes systematically and quantitatively to reflect the course of PCa, yet has remained undefined due to a lack of tools that can define collagen protein structure. Here we use a novel collagen-targeting proteomics approach to investigate zonal regulation of collagen-type proteins in PCa prostatectomies. METHODS: Prostatectomies from nine patients were divided into zones containing 0%, 5%, 20%, 70% to 80% glandular tissue and 0%, 5%, 25%, 70% by mass of PCa tumor following the McNeal model. Tissue sections from zones were graded by a pathologist for Gleason score, percent tumor present, percent prostatic intraepithelial neoplasia and/or inflammation (INF). High-resolution accurate mass collagen targeting proteomics was done on a select subset of tissue sections from patient-matched tumor or nontumor zones. Imaging mass spectrometry was used to investigate collagen-type regulation corresponding to pathologist-defined regions. RESULTS: Complex collagen proteomes were detected from all zones. COL17A and COL27A increased in zones of INF compared with zones with tumor present. COL3A1, COL4A5, and COL8A2 consistently increased in zones with tumor content, independent of tumor size. Collagen hydroxylation of proline (HYP) was altered in tumor zones compared with zones with INF and no tumor. COL3A1 and COL5A1 showed significant changes in HYP peptide ratios within tumor compared with zones of INF (2.59 ± 0.29, P value: .015; 3.75 ± 0.96 P value .036, respectively). By imaging mass spectrometry COL3A1 showed defined localization and regulation to tumor pathology. COL1A1 and COL1A2 showed gradient regulation corresponding to PCa pathology across zones. Pathologist-defined tumor regions showed significant increases in COL1A1 HYP modifications compared with COL1A2 HYP modifications. Certain COL1A1 and COL1A2 peptides could discriminate between pathologist-defined tumor and inflammatory regions. CONCLUSIONS: Site-specific posttranslational regulation of collagen structure by proline hydroxylation may be involved in reactive stroma associated with PCa progression. Translational and posttranslational regulation of collagen protein structure has potential for new markers to understand PCa progression and outcomes.


Assuntos
Colágeno/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Processamento de Proteína Pós-Traducional , Idoso , Sequência de Aminoácidos , Autoantígenos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Colágeno Tipo IV/metabolismo , Colágeno Tipo VIII/metabolismo , Progressão da Doença , Colágenos Fibrilares/metabolismo , Humanos , Hidroxilação , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Colágenos não Fibrilares , Prolina/metabolismo , Próstata/metabolismo , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos
14.
PLoS One ; 15(7): e0236505, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701970

RESUMO

Multidrug resistance prompts the search for new sources of antibiotics with new targets at bacteria cell. To investigate the antibacterial activity of Cinnamomum cassia L. essential oil (CCeo) alone and in combination with antibiotics against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens. The antimicrobial susceptibility of the strains was determined by Vitek® 2 and confirmed by MALDI-TOF/TOF. The antibacterial activity of CCeo and its synergism with antibiotics was determined using agar disk diffusion, broth microdilution, time-kill, and checkboard methods. The integrity of the bacterial cell membrane in S. marcescens was monitored by protein leakage assay. CCeo exhibited inhibitory effects with MIC = 281.25 µg.mL-1. The association between CCeo and polymyxin B showed a decrease in terms of viable cell counts on survival curves over time after a 4 hour-treatment with a FIC index value of 0.006. Protein leakage was observed with increasing concentrations for CCeo and CCeo + polymyxin B treatments. CCeo showed antibacterial activity against the studied strains. When associated with polymyxin B, a synergistic effect was able to inhibit bacterial growth rapidly and consistently, making it a potential candidate for the development of an alternative treatment and drug delivery system for carbapenemase-producing strains.


Assuntos
Infecções por Klebsiella/tratamento farmacológico , Óleos Voláteis/farmacologia , Polimixina B/farmacologia , Infecções por Serratia/tratamento farmacológico , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Cinnamomum aromaticum/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Humanos , Infecções por Klebsiella/genética , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/patogenicidade , Infecções por Serratia/genética , Infecções por Serratia/microbiologia , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/patogenicidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , beta-Lactamases/genética
15.
Dermatol Online J ; 26(4)2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32621684

RESUMO

Non-pigmented rapidly growing mycobacteria are nontuberculous mycobacteria (NTM) capable of producing disease. We report a case of tattoo-associated NTM infection with a novel species: Mycobacterium mageritense. A 48-year-old man presented with a two-week history of a papulopustular eruption on the shaded areas of a tattoo that had been placed five weeks prior while in the Philippines. Histopathology from punch biopsies revealed suppurative granulomatous dermatitis with acid fast bacilli present. Subsequent matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometer identified the bacteria as Mycobacterium margeritense. After consultation with infectious disease specialists and culture susceptibilities, the patient was treated with three months of dual antibiotic therapy with minocycline and moxifloxacin. The patient experienced a slow but complete resolution of clinical skin findings after the course of treatment. Since discovery in 1997, M. mageritense infection has been demonstrated in a wide spectrum of disease, predominantly skin and soft tissue infections. The species has not been previously implicated in tattoo-associated NTM infections. M. mageritense should be considered as a specific type of mycobacteria in the differential diagnosis for tattoo-associated NTM infections owing to differences in antibiotic susceptibilities compared to other NTM species.


Assuntos
Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Tatuagem/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Pele/microbiologia , Pele/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
PLoS One ; 15(6): e0231679, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32559193

RESUMO

The envelope glycoprotein (Env) of the human immunodeficiency virus (HIV), has been the primary target for the development of a protective vaccine against infection. The extensive N-linked glycosylation on Env is an important consideration as it may affect efficacy, stability, and expression yields. The expression host has been shown to influence the extent and type of glycosylation that decorates the protein target. Here, we report the glycosylation profile of the candidate subtype C immunogen CO6980v0c22 gp145, which is currently in Phase I clinical trials, produced in two different host cells: CHO-K1 and Expi293F. The amino acid sequence for both glycoproteins was confirmed to be identical by peptide mass fingerprinting. However, the isoelectric point of the proteins differed; 4.5-5.5 and 6.0-7.0 for gp145 produced in CHO-K1 and Expi293F, respectively. These differences in pI were eliminated by enzymatic treatment with sialidase, indicating a large difference in the incorporation of sialic acid between hosts. This dramatic difference in the number of sialylated glycans between hosts was confirmed by analysis of PNGase F-released glycans using MALDI-ToF MS. These differences in glycosylation, however, did not greatly translate into differences in antibody recognition. Biosensor assays showed that gp145 produced in CHO-K1 had similar affinity toward the broadly neutralizing antibodies, 2G12 and PG16, as the gp145 produced in Expi293F. Additionally, both immunogens showed the same reactivity against plasma of HIV-infected patients. Taken together, these results support the notion that there are sizeable differences in the glycosylation of Env depending on the expression host. How these differences translate to vaccine efficacy remains unknown.


Assuntos
Glicopeptídeos/análise , Anticorpos Anti-HIV/imunologia , HIV-1/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Adulto , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Reações Antígeno-Anticorpo , Células CHO , Cricetinae , Cricetulus , Feminino , Glicosilação , Células HEK293 , Humanos , Pessoa de Meia-Idade , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
17.
PLoS One ; 15(6): e0232610, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497137

RESUMO

Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery. However, in most cases, the causative agent cannot be identified, which is partly due to the limited spectrum of pathogens covered by current diagnostics based on nucleic acid amplification. Therefore, in this study, we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. From April to July 2017, 32 hospitalized children with severe nonresponding pneumonia in Shenzhen Children's Hospital were included in this study. Blood tests were conducted immediately after hospitalization to assess cell counts and inflammatory markers, oropharyngeal swabs were collected to identify common pathogens by qPCR and culture. After bronchoscopy, bronchoalveolar lavage fluid (BALF) samples were collected for further pathogen identification using standardized diagnostic tests and mNGS. Blood tests were normal in 3 of the 32 children. In 9 oropharyngeal swabs, bacterial pathogens were detected, in 5 of these Mycoplasma pneumoniae was detected. Adenovirus was detected in 5 BALF samples, using the Direct Immunofluorescence Assay (DFA). In 15 cases, no common pathogens were found in BALF samples, using the current standard diagnostic tests, while in all 32 BALFs, pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus and bocavirus. This study shows that, with mNGS, the sensitivity of detection of the causative pathogens in children with severe nonresponding pneumonia is significantly improved. In addition, mNGS gives more strain specific information, helps to identify new pathogens and could potentially help to trace and control outbreaks. In this study, we have shown that it is possible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.


Assuntos
Coinfecção , Metagenômica/métodos , Pneumonia Bacteriana/diagnóstico , Pneumonia Viral/diagnóstico , Contagem de Células Sanguíneas , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , Criança , Pré-Escolar , China/epidemiologia , Coinfecção/microbiologia , Coinfecção/virologia , DNA Bacteriano/análise , DNA Viral/análise , Feminino , Técnica Direta de Fluorescência para Anticorpo , Humanos , Lactente , Pacientes Internados , Masculino , Metagenoma , Orofaringe/microbiologia , Orofaringe/virologia , Pneumonia Bacteriana/epidemiologia , Pneumonia Bacteriana/microbiologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Estudo de Prova de Conceito , RNA Viral/análise , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
J Breath Res ; 14(4): 041001, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32531777

RESUMO

The COVID-19 pandemic has highlighted the importance of rapid, cost effective, accurate, and non-invasive testing for viral infections. Volatile compounds (VCs) have been suggested for several decades as fulfilling these criteria. However currently very little work has been done in trying to diagnose viral infections using VCs. Much of the work carried out to date involves the differentiation of bacterial and viral sources of infection and often the detection of bacterial and viral co-infection. However, this has usually been done in vitro and very little work has involved the use of human participants. Viruses hijack the host cell metabolism and do not produce their own metabolites so identifying virus specific VCs is at best a challenging task. However, there are proteins and lipids that are potential candidates as markers of viral infection. The current understanding is that host cell glycolysis is upregulated under viral infection to increase the available energy for viral replication. There is some evidence that viral infection leads to the increase of production of fatty acids, alkanes, and alkanes related products. For instance, 2,3-butandione, aldehydes, 2,8-dimethyl-undecane and n-propyl acetate have all been correlated with viral infection. Currently, the literature points to markers of oxidative stress (e.g. nitric oxide, aldehydes etc) being the most useful in the determination of viral infection. The issue, however, is that there are also many other conditions that can lead to oxidative stress markers being produced. In this review a range of (mainly mass spectrometric) methods are discussed for viral detection in breath, including breath condensate. Currently MALDI-ToF-MS is likely to be the preferred method for the identification of viral strains and variants of those strains, however it is limited by its need for the viral strains to have been sequenced and logged in a database.


Assuntos
Testes Respiratórios/métodos , Viroses/diagnóstico , Aldeídos/metabolismo , Animais , Betacoronavirus , Biomarcadores/metabolismo , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Hepatite B/diagnóstico , Hepatite B/metabolismo , Humanos , Influenza Humana/diagnóstico , Influenza Humana/metabolismo , Espectrometria de Massas , Óxido Nítrico/metabolismo , Infecções por Orthomyxoviridae/diagnóstico , Infecções por Orthomyxoviridae/metabolismo , Estresse Oxidativo , Pandemias , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/metabolismo , Pneumonia Viral/diagnóstico , Pneumonia Viral/metabolismo , Infecções por Rotavirus/diagnóstico , Infecções por Rotavirus/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Viroses/metabolismo , Vírus
19.
PLoS One ; 15(6): e0234989, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32598367

RESUMO

Alterations in glycosylation are seen in many types of cancer, including colorectal cancer (CRC). Glycans, the sugar moieties of glycoconjugates, are involved in many important functions relevant to cancer and can be of value as biomarkers. In this study, we have used mass spectrometry to analyze the N-glycan profiles of 35 CRC tissue samples and 10 healthy tissue samples from non-CRC patients who underwent operations for other reasons. The tumor samples were divided into groups depending on tumor location (right or left colon) and stage (II or III), while the healthy samples were divided into right or left colon. The levels of neutral and acidic N-glycan compositions and glycan classes were analyzed in a total of ten different groups. Surprisingly, there were no significant differences in glycan levels when all right- and left-sided CRC samples were compared, and few differences (such as in the abundance of the neutral N-glycan H3N5) were seen when the samples were divided according to both location and stage. Multiple significant differences were found in the levels of glycans and glycan classes when stage II and III samples were compared, and these glycans could be of value as candidates for new markers of cancer progression. In order to validate our findings, we analyzed healthy tissue samples from the right and left colon and found no significant differences in the levels of any of the glycans analyzed, confirming that our findings when comparing CRC samples from the right and left colon are not due to normal variations in the levels of glycans between the healthy right and left colon. Additionally, the levels of the acidic glycans H4N3F1P1, H5N4F1P1, and S1H5N4F1 were found to change in a cancer-specific but colon location-nonspecific manner, indicating that CRC affects glycan levels in similar ways regardless of tumor location.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/metabolismo , Glicômica , Polissacarídeos/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Glicosilação , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Adulto Jovem
20.
J Med Microbiol ; 69(8): 1105-1113, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32597748

RESUMO

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.


Assuntos
Burkholderia cepacia/isolamento & purificação , Burkholderia gladioli/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Técnicas de Tipagem Bacteriana/métodos , Burkholderia cepacia/classificação , Burkholderia gladioli/classificação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Análise de Fourier , Humanos , Tipagem de Sequências Multilocus , Filogenia , Recombinases Rec A/genética , Alinhamento de Sequência
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