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1.
J Immunol ; 204(5): 1263-1273, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31932496

RESUMO

The poly-γ-glutamic acid (PGA) capsule produced by Bacillus anthracis is composed entirely of d-isomer glutamic acid, whereas nonpathogenic Bacillus species produce mixed d-, l-isomer PGAs. To determine if B. anthracis PGA confers a pathogenic advantage over other PGAs, we compared the responses of human innate immune cells to B. anthracis PGA and PGAs from nonpathogenic B. subtilis subsp. chungkookjang and B. licheniformis Monocytes and immature dendritic cells (iDCs) responded differentially to the PGAs, with B. anthracis PGA being least stimulatory and B. licheniformis PGA most stimulatory. All three elicited IL-8 and IL-6 from monocytes, but B. subtilis PGA also elicited IL-10 and TNF-α, whereas B. licheniformis PGA elicited all those plus IL-1ß. Similarly, all three PGAs elicited IL-8 from iDCs, but B. subtilis PGA also elicited IL-6, and B. licheniformis PGA elicited those plus IL-12p70, IL-10, IL-1ß, and TNF-α. Only B. licheniformis PGA induced dendritic cell maturation. TLR assays also yielded differential results. B. subtilis PGA and B. licheniformis PGA both elicited more TLR2 signal than B. anthracis PGA, but only responses to B. subtilis PGA were affected by a TLR6 neutralizing Ab. B. licheniformis PGA elicited more TLR4 signal than B. anthracis PGA, whereas B. subtilis PGA elicited none. B. anthracis PGA persisted longer in high m.w. form in monocyte and iDC cultures than the other PGAs. Reducing the m.w. of B. anthracis PGA reduced monocytes' cytokine responses. We conclude that B. anthracis PGA is recognized less effectively by innate immune cells than PGAs from nonpathogenic Bacillus species, resulting in failure to induce a robust host response, which may contribute to anthrax pathogenesis.

3.
N Engl J Med ; 382(8): 697-705, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31860793

RESUMO

BACKGROUND: The causative agents for the current national outbreak of electronic-cigarette, or vaping, product use-associated lung injury (EVALI) have not been established. Detection of toxicants in bronchoalveolar-lavage (BAL) fluid from patients with EVALI can provide direct information on exposure within the lung. METHODS: BAL fluids were collected from 51 patients with EVALI in 16 states and from 99 healthy participants who were part of an ongoing study of smoking involving nonsmokers, exclusive users of e-cigarettes or vaping products, and exclusive cigarette smokers that was initiated in 2015. Using the BAL fluid, we performed isotope dilution mass spectrometry to measure several priority toxicants: vitamin E acetate, plant oils, medium-chain triglyceride oil, coconut oil, petroleum distillates, and diluent terpenes. RESULTS: State and local health departments assigned EVALI case status as confirmed for 25 patients and as probable for 26 patients. Vitamin E acetate was identified in BAL fluid obtained from 48 of 51 case patients (94%) in 16 states but not in such fluid obtained from the healthy comparator group. No other priority toxicants were found in BAL fluid from the case patients or the comparator group, except for coconut oil and limonene, which were found in 1 patient each. Among the case patients for whom laboratory or epidemiologic data were available, 47 of 50 (94%) had detectable tetrahydrocannabinol (THC) or its metabolites in BAL fluid or had reported vaping THC products in the 90 days before the onset of illness. Nicotine or its metabolites were detected in 30 of 47 of the case patients (64%). CONCLUSIONS: Vitamin E acetate was associated with EVALI in a convenience sample of 51 patients in 16 states across the United States. (Funded by the National Cancer Institute and others.).


Assuntos
Lesão Pulmonar Aguda/patologia , Líquido da Lavagem Broncoalveolar/química , Sistemas Eletrônicos de Liberação de Nicotina , Vaping/efeitos adversos , Vitamina E/análise , Lesão Pulmonar Aguda/etiologia , Adolescente , Adulto , Idoso , Fumar Cigarros , Óleo de Coco/análise , Feminino , Humanos , Limoneno/análise , Masculino , Pessoa de Meia-Idade , Estados Unidos , Adulto Jovem
4.
Sci Rep ; 9(1): 15876, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685918

RESUMO

Cystic hydatid disease (CHD) is a worldwide neglected zoonotic disease caused by Echinococcus granulosus. The parasite is well adapted to its host by producing protective molecules that modulate host immune response. An unexplored issue associated with the parasite's persistence in its host is how the organism can survive the oxidative stress resulting from parasite endogenous metabolism and host defenses. Here, we used hydrogen peroxide (H2O2) to induce oxidative stress in E. granulosus protoescoleces (PSCs) to identify molecular pathways and antioxidant responses during H2O2 exposure. Using proteomics, we identified 550 unique proteins; including 474 in H2O2-exposed PSCs (H-PSCs) samples and 515 in non-exposed PSCs (C-PSCs) samples. Larger amounts of antioxidant proteins, including GSTs and novel carbonyl detoxifying enzymes, such as aldo-keto reductase and carbonyl reductase, were detected after H2O2 exposure. Increased concentrations of caspase-3 and cathepsin-D proteases and components of the 26S proteasome were also detected in H-PSCs. Reduction of lamin-B and other caspase-substrate, such as filamin, in H-PSCs suggested that molecular events related to early apoptosis were also induced. We present data that describe proteins expressed in response to oxidative stress in a metazoan parasite, including novel antioxidant enzymes and targets with potential application to treatment and prevention of CHD.

5.
MMWR Morb Mortal Wkly Rep ; 68(45): 1040-1041, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31725707

RESUMO

CDC, the Food and Drug Administration (FDA), state and local health departments, and multiple public health and clinical partners are investigating a national outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Based on data collected as of October 15, 2019, 86% of 867 EVALI patients reported using tetrahydrocannabinol (THC)-containing products in the 3 months preceding symptom onset (1). Analyses of THC-containing product samples by FDA and state public health laboratories have identified potentially harmful constituents in these products, such as vitamin E acetate, medium chain triglyceride oil (MCT oil), and other lipids (2,3) (personal communication, D.T. Heitkemper, FDA Forensic Chemistry Center, November 2019). Vitamin E acetate, in particular, might be used as an additive in the production of e-cigarette, or vaping, products; it also can be used as a thickening agent in THC products (4). Inhalation of vitamin E acetate might impair lung function (5-7).


Assuntos
Líquido da Lavagem Broncoalveolar/química , Surtos de Doenças , Lesão Pulmonar/epidemiologia , Vaping/efeitos adversos , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estados Unidos/epidemiologia , Adulto Jovem
6.
Microb Pathog ; 137: 103717, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31494300

RESUMO

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), a widespread disease that causes major economic losses to the pig industry. The swine host response plays an important role in the outcome of M. hyopneumoniae infections. The whole proteome of newborn pig trachea (NPTr) epithelial cells infected with the M. hyopneumoniae pathogenic strain 7448 was analyzed using an LC-MS/MS approach to shed light on intracellular processes triggered in response to the pathogen. Overall, 853 swine protein species were identified, 156 of which were differentially represented in response to M. hyopneumoniae 7448 infection in comparison with non-infected control cells. These differentially represented proteins were categorized by function. Fifty-seven of them were assigned to the immune system and/or response to stimulus functional subcategories. Comparative expression analysis of these immune-related proteins in NPTr cells infected with attenuated or non-pathogenic mycoplasmas (M. hyopneumoniae J strain and M. flocculare, respectively) revealed proteins whose abundance was altered only in response to the pathogenic M. hyopneumoniae 7448 strain. Among these proteins, calcium homeostasis and endoplasmic reticulum stress-related biomarkers were detected, providing evidence of molecular mechanisms that might lead to swine cell apoptosis.

7.
J Anal Toxicol ; 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31287544

RESUMO

Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism-a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.

8.
Anal Bioanal Chem ; 411(21): 5489-5497, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31172236

RESUMO

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A-G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution.


Assuntos
Toxinas Botulínicas/análise , Peptídeos/química , Humanos , Limite de Detecção
9.
Int J Syst Evol Microbiol ; 69(8): 2268-2276, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31125302

RESUMO

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.


Assuntos
Enterococcaceae/classificação , Pé/microbiologia , Filogenia , Carne Vermelha/microbiologia , Ferimentos e Lesões/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Enterococcaceae/isolamento & purificação , Ácidos Graxos/química , Genes Bacterianos , Humanos , Masculino , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
10.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1118-1119: 137-147, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31035135

RESUMO

Progress toward better diagnosis and treatment of lipid metabolism-related diseases requires high throughput approaches for multiplexed quantitative analysis of structurally diverse lipids, including phospholipids (PLs). This work demonstrates a simplified "one-pot" phospholipid extraction protocol, as an alternative to conventional liquid-liquid extraction. Performed in a 96-well format, the extraction was coupled with high throughput UPLC and multiplexed tandem mass spectrometry (MS/MS) detection, allowing non-targeted quantification of phosphatidylcholines (PC), sphingomyelins (SM), lysophosphatidylcholines (LPC), phosphatidylethanolamines (PE), and phosphatidylinositols (PI). Using 50 µL aliquots of serum samples from 110 individuals, lipoproteins were fractionated by size, and analyzed for phospholipids and non-polar lipids including free cholesterol (FC), cholesteryl esters (CEs) and triglycerides (TGs). Analysis of serum samples with wide range of Total-TG levels showed significant differences in PL composition. The correlations of molar ratios in lipoprotein size fractions, SM/PL with FC/PL, PE/PL with TG/CE, and PE/PL with PI/PL, demonstrate the applicability of the method for quantitative composition analysis of high, low and very-low density lipoproteins (HDL, LDL and VLDL), and characterization of lipid metabolism related disease states.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Dislipidemias/sangue , Dislipidemias/diagnóstico , Fosfolipídeos/sangue , Espectrometria de Massas em Tandem/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fosfolipídeos/isolamento & purificação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
11.
J Proteomics ; 199: 67-76, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30862566

RESUMO

Mycoplasma hyopneumoniae is a respiratory pathogen, causing porcine enzootic pneumonia. To survive in the porcine respiratory tract, M. hyopneumoniae must cope with both oxidative and heat stress imposed by the host. To get insights into M. hyopneumoniae stress responses and pathogenicity mechanisms, the protein profiles of two M. hyopneumoniae strains, pathogenic 7448 strain and non-pathogenic strain J, were surveyed under oxidative (OS) or heat (HS) stress. M. hyopneumoniae strains were submitted to OS (0.5% hydrogen peroxide) or HS (temperature shifts to 42 °C) conditions and protein profiling was carried out by LC-MS/MS and label-free quantitative analyses. Data are available via ProteomeXchange with identifier PXD012742. Qualitative and quantitative differences involving 40-60 M. hyopneumoniae proteins were observed for both strains when comparing bacteria exposed to OS or HS to non-treated controls. However, no differences in abundance were found in proteins classically related to stress responses, as peroxidases and chaperones, suggesting that these proteins would be constitutively present in both strains in the tested conditions. Interestingly, under stress conditions, more virulence-related proteins were detected in M. hyopneumoniae 7448 differentially represented proteins than in M. hyopneumoniae J, suggesting that stress may trigger a differential response of the corresponding genes, shared by both strains.

12.
Anal Bioanal Chem ; 411(12): 2493-2509, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30911800

RESUMO

Inhalation of Bacillus anthracis spores can cause a rapidly progressing fatal infection. B. anthracis secretes three protein toxins: lethal factor (LF), edema factor (EF), and protective antigen (PA). EF and LF may circulate as free or PA-bound forms. Both free EF (EF) and PA-bound-EF (ETx) have adenylyl cyclase activity converting ATP to cAMP. We developed an adenylyl cyclase activity-based method for detecting and quantifying total EF (EF+ETx) in plasma. The three-step method includes magnetic immunocapture with monoclonal antibodies, reaction with ATP generating cAMP, and quantification of cAMP by isotope-dilution HPLC-MS/MS. Total EF was quantified from 5PL regression of cAMP vs ETx concentration. The detection limit was 20 fg/mL (225 zeptomoles/mL for the 89 kDa protein). Relative standard deviations for controls with 0.3, 6.0, and 90 pg/mL were 11.7-16.6% with 91.2-99.5% accuracy. The method demonstrated 100% specificity in 238 human serum/plasma samples collected from unexposed healthy individuals, and 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Similar to LF, the kinetics of EF over the course of infection were triphasic, with an initial rise (phase-1), decline (phase-2), and final rapid rise (phase-3). EF levels were ~ 2-4 orders of magnitude lower than LF during phase-1 and phase-2 and only ~ 6-fold lower at death/euthanasia. Analysis of EF improves early diagnosis and adds to our understanding of anthrax toxemia throughout infection. The LF/EF ratio may also indicate the stage of infection and need for advanced treatments.


Assuntos
Antraz/patologia , Antígenos de Bactérias/sangue , Bacillus anthracis/patogenicidade , Toxinas Bacterianas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Infecções Respiratórias/patologia , Espectrometria de Massas em Tandem/métodos , Toxemia/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antraz/sangue , Estudos de Casos e Controles , AMP Cíclico/biossíntese , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Humanos , Limite de Detecção , Macaca mulatta , Reação em Cadeia da Polimerase , Infecções Respiratórias/sangue , Toxemia/sangue , Toxemia/microbiologia
13.
Analyst ; 144(7): 2264-2274, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30810119

RESUMO

Anthrax protective antigen (83 kDa, PA83) is an essential component of two major binary toxins produced by Bacillus anthracis, lethal toxin (LTx) and edema toxin (ETx). During infection, LTx and ETx contribute to immune collapse, endothelial dysfunction, hemorrhage and high mortality. Following protease cleavage on cell receptors or in circulation, the 20 kDa (PA20) N-terminus is released, activating the 63 kDa (PA63) form which binds lethal factor (LF) and edema factor (EF), facilitating their entry into their cellular targets. Several ELISA-based PA methods previously developed are primarily qualitative or semi-quantitative. Here, we combined protein immunocapture, tryptic digestion and isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS), to develop a highly selective and sensitive method for detection and accurate quantification of total-PA (PA83 + PA63) and PA83. Two tryptic peptides in the 63 kDa region measure total-PA and three in the 20 kDa region measure PA83 alone. Detection limits range from 1.3-2.9 ng mL-1 PA in 100 µL of plasma. Spiked recovery experiments with combinations of PA83, PA63, LF and EF in plasma showed that PA63 and PA83 were quantified accurately against the PA83 standard and that LF and EF did not interfere with accuracy. Applied to a study of inhalation anthrax in rhesus macaques, total-PA suggested triphasic kinetics, similar to that previously observed for LF and EF. This study is the first to report circulating PA83 in inhalation anthrax, typically at less than 4% of the levels of PA63, providing the first evidence that activated PA63 is the primary form of PA throughout infection.


Assuntos
Antígenos de Bactérias/sangue , Bacillus anthracis/imunologia , Toxinas Bacterianas/sangue , Imunoensaio/métodos , Limite de Detecção , Espectrometria de Massas , Animais , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Macaca mulatta
14.
J Proteomics ; 192: 147-159, 2019 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-30176387

RESUMO

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar. However, M. hyopneumoniae causes porcine enzootic pneumonia, while M. flocculare is a commensal bacterium. M. hyopneumoniae and M. flocculare do not penetrate their host cells, and secreted proteins are important for bacterium-host interplay. Thus, the secretomes of a swine trachea cell line (NPTr) infected with M. hyopneumoniae 7448 (a pathogenic strain), M. hyopneumoniae J (a non-pathogenic strain) and M. flocculare were compared to shed light in bacterium-host interactions. Medium from the cultures was collected, and secreted proteins were identified by a LC-MS/MS. Overall numbers of identified host and bacterial proteins were, respectively, 488 and 58, for NPTr/M. hyopneumoniae 7448; 371 and 67, for NPTr/M. hyopneumoniae J; and 203 and 81, for NPTr/M. flocculare. The swine cells revealed different secretion profiles in response to the infection with each M. hyopneumoniae strain or with M. flocculare. DAMPs and extracellular proteasome proteins, secreted in response to cell injury and death, were secreted by NPTr cells infected with M. hyopneumoniae 7448. All three mycoplasmas secreted virulence factors during NPTr infection, but M. hyopneumoniae 7448 secreted higher number of adhesins and hypothetical proteins, that may be related with pathogenicity. SIGNIFICANCE: The enzootic pneumonia caused by mycoplasmas of swine respiratory tract has economic loss consequences in pig industry due to antibiotic costs and pig weight loss. However, some genetically similar mycoplasmas are pathogenic while others, such as Mycoplasma hyopneumoniae and Mycoplasma flocculare, are non-pathogenic. Here, we conducted an infection assay between swine cells and pathogenic and non-pathogenic mycoplasmas to decipher secreted proteins during host-pathogen interaction. Mycoplasma response to cell infection was also observed. Our study provided new insights on secretion profile of swine cells in response to the infection with pathogenic and non-pathogenic mycoplasmas. It was possible to observe that pathogenic M. hyopneumoniae 7448 secreted known virulence factors and swine cells responded by inducing cell death. Otherwise, M. hyopneumoniae J and M. flocculare, non-pathogenic mycoplasmas, secreted a different profile of virulence factors in response to swine cells. Consequently, swine cells altered their secretome profile, but the changes were not sufficient to cause disease.


Assuntos
Proteínas de Bactérias/metabolismo , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma/metabolismo , Pneumonia Suína Micoplasmática/metabolismo , Proteoma/metabolismo , Suínos/microbiologia , Traqueia/microbiologia , Animais , Linhagem Celular , Pneumonia Suína Micoplasmática/microbiologia
15.
Methods Mol Biol ; 1871: 295-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30276747

RESUMO

Protein digestion coupled to liquid chromatography and tandem mass spectrometry (LC-MS/MS) detection enables multiplexed quantification of proteins in complex biological matrices. However, the reproducibility of enzymatic digestion of proteins to produce proteotypic target peptides is a major limiting factor of assay precision. Online digestion using immobilized trypsin addresses this problem through precise control of digestion conditions and time. Because online digestion is typically for a short time, the potential for peptide degradation, a major source of measurement bias, is significantly reduced. Online proteolysis requires minimal sample preparation and is easily coupled to LC-MS/MS systems, further reducing potential method variability. We describe herein a method optimized for the multiplexed quantification of several apolipoproteins in human serum using on-column digestion. We highlight key features of the method that enhance assay accuracy and precision. These include the use of value-assigned serum as calibrators and stable isotope-labeled (SIL) peptide analogs as internal standards. We also comment on practical aspects of column switching valve design, instrument maintenance, tandem mass spectrometry data acquisition, and data processing.


Assuntos
Cromatografia Líquida , Proteínas , Espectrometria de Massas em Tandem , Tripsina , Apolipoproteínas/análise , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Proteínas Sanguíneas/química , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Análise de Dados , Enzimas Imobilizadas , Humanos , Peptídeos/química , Proteínas/análise , Proteínas/química , Proteólise , Software , Espectrometria de Massas em Tandem/métodos , Tripsina/química
16.
Vaccine ; 36(41): 6144-6151, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194004

RESUMO

Influenza vaccines are the most effective intervention to prevent the substantial public health burden of seasonal and pandemic influenza. Hemagglutinin (HA), as the main antigen in inactivated influenza vaccines (IIVs), elicits functional neutralizing antibodies and largely determines IIV effectiveness. HA potency has been evaluated by single-radial immunodiffusion (SRID), the standard in vitro potency assay for IIVs, to predict vaccine immunogenicity with a correlation to protective efficacy. We previously reported that limited trypsin digestion (LTD) selectively degraded stressed HA, so that an otherwise conformationally insensitive biophysical quantification technique could specifically quantify trypsin-resistant, immunologically active HA. Here, we demonstrate that isotope dilution mass spectrometry (IDMS), a method capable of quantifying the absolute HA concentration without reference antigen use, can be further expanded by adding LTD followed with precipitation to selectively quantify the active HA. We test the LTD-IDMS assay on H7N9 vaccines stressed by low pH, raised temperature, or freeze/thaw cycles. This method, unlike SRID, has no requirement for strain-specific reference antigens or antibodies and can generate potency values that correlate with SRID. Thus, LTD-IDMS is a promising alternative in vitro potency assay for influenza vaccines to complement and potentially replace SRID in a pandemic when strain specific reagents may not be readily available.


Assuntos
Subtipo H7N9 do Vírus da Influenza A/imunologia , Subtipo H7N9 do Vírus da Influenza A/patogenicidade , Vacinas contra Influenza/uso terapêutico , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Hemaglutininas/metabolismo , Humanos , Marcação por Isótopo , Espectrometria de Massas
17.
Virulence ; 9(1): 1230-1246, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30027802

RESUMO

Mycoplasma hyopneumoniae and Mycoplasma flocculare are genetically similar bacteria, which coinhabit the porcine respiratory tract. These mycoplasmas share most of the known virulence factors, but, while M. hyopneumoniae causes porcine enzootic pneumonia (PEP), M. flocculare is a commensal species. To identify potential PEP determinants and provide novel insights on mycoplasma-host interactions, the whole cell proteomes of two M. hyopneumoniae strains, one pathogenic (7448) and other non-pathogenic (J), and M. flocculare were compared. A cell fractioning approach combined with mass spectrometry (LC-MS/MS) proteomics was used to analyze cytoplasmic and surface-enriched protein fractions. Average detection of ~ 50% of the predicted proteomes of M. hyopneumoniae 7448 and J, and M. flocculare was achieved. Many of the identified proteins were differentially represented in M. hyopneumoniae 7448 in comparison to M. hyopneumoniae J and M. flocculare, including potential PEP determinants, such as adhesins, proteases, and redox-balancing proteins, among others. The LC-MS/MS data also provided experimental validation for several genes previously regarded as hypothetical for all analyzed mycoplasmas, including some coding for proteins bearing virulence-related functional domains. The comprehensive proteome profiling of two M. hyopneumoniae strains and M. flocculare provided tens of novel candidates to PEP determinants or virulence factors, beyond those classically described.


Assuntos
Interações entre Hospedeiro e Microrganismos , Mycoplasma hyopneumoniae/metabolismo , Mycoplasma/metabolismo , Pneumonia Suína Micoplasmática/microbiologia , Proteoma/metabolismo , Adesinas Bacterianas/análise , Animais , Proteínas de Bactérias/análise , Espectrometria de Massas , Mycoplasma hyopneumoniae/patogenicidade , Peptídeo Hidrolases/análise , Especificidade da Espécie , Suínos , Fatores de Virulência
18.
PLoS One ; 13(4): e0194797, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29634782

RESUMO

Lipoproteins are complex molecular assemblies that are key participants in the intricate cascade of extracellular lipid metabolism with important consequences in the formation of atherosclerotic lesions and the development of cardiovascular disease. Multiplexed mass spectrometry (MS) techniques have substantially improved the ability to characterize the composition of lipoproteins. However, these advanced MS techniques are limited by traditional pre-analytical fractionation techniques that compromise the structural integrity of lipoprotein particles during separation from serum or plasma. In this work, we applied a highly effective and gentle hydrodynamic size based fractionation technique, asymmetric flow field-flow fractionation (AF4), and integrated it into a comprehensive tandem mass spectrometry based workflow that was used for the measurement of apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E), free cholesterol (FC), cholesterol esters (CE), triglycerides (TG), and phospholipids (PL) (phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC)). Hydrodynamic size in each of 40 size fractions separated by AF4 was measured by dynamic light scattering. Measuring all major lipids and apolipoproteins in each size fraction and in the whole serum, using total of 0.1 ml, allowed the volumetric calculation of lipoprotein particle numbers and expression of composition in molar analyte per particle number ratios. Measurements in 110 serum samples showed substantive differences between size fractions of HDL and LDL. Lipoprotein composition within size fractions was expressed in molar ratios of analytes (A-I/A-II, C-II/C-I, C-II/C-III. E/C-III, FC/PL, SM/PL, PE/PL, and PI/PL), showing differences in sample categories with combinations of normal and high levels of Total-C and/or Total-TG. The agreement with previous studies indirectly validates the AF4-LC-MS/MS approach and demonstrates the potential of this workflow for characterization of lipoprotein composition in clinical studies using small volumes of archived frozen samples.


Assuntos
Apolipoproteínas/sangue , Cromatografia Líquida/métodos , Fracionamento por Campo e Fluxo/métodos , Lipídeos/sangue , Lipoproteínas/sangue , Espectrometria de Massas em Tandem/métodos , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Análise Química do Sangue/métodos , Calibragem , Colesterol/química , Humanos , Luz , Modelos Estatísticos , Tamanho da Partícula , Controle de Qualidade , Espalhamento de Radiação , Fluxo de Trabalho
19.
J Proteomics ; 175: 127-135, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29317356

RESUMO

Mesocestoides corti is a widely used model for the study of cestode biology, and its transition from the larval tetrathyridium (TT) stage to the strobilated, adult worm (ST) stage can be induced and followed in vitro. Here, a proteomic approach was used to describe and compare M. corti TT and ST protein repertories. Overall, 571 proteins were identified, 238 proteins in TT samples and 333 proteins in ST samples. Among the identified proteins, 207 proteins were shared by TTs and STs, while 157 were stage-specific, being 31 exclusive from TTs, and 126 from STs. Functional annotation revealed fundamental metabolic differences between the TT and the ST stages. TTs perform functions related mainly to basic metabolism, responsible for growth and vegetative development by asexual reproduction. STs, in contrast, perform a wider range of functions, including macromolecule biosynthetic processes, gene expression and control pathways, which may be associated to its proglottization/segmentation, sexual differentiation and more complex physiology. Furthermore, the generated results provided an extensive list of cestode proteins of interest for functional studies in M. corti. Many of these proteins are novel candidate diagnostic antigens, and/or potential targets for the development of new and more effective antihelminthic drugs. BIOLOGICAL SIGNIFICANCE: Cestodiases are parasitic diseases with serious impact on human and animal health. Efforts to develop more effective strategies for diagnosis, treatment or control of cestodiases are impaired by the still limited knowledge on many aspects of cestode biology, including the complex developmental processes that occur in the life cycles of these parasites. Mesocestoides corti is a good experimental model to study the transition from the larval to the adult stage, called strobilation, which occur in typical cestode life-cycles. The performed proteomics approach provided large-scale identification and quantification of M. corti proteins. Many stage-specific or differentially expressed proteins were detected in the larval tetrathyridium (TT) stage and in the strobilated, adult worm (ST) stage. Functional comparative analyses of the described protein repertoires shed light on function and processes associated to specific features of both stages, such as less differentiation and asexual reproduction in TTs, and proglottization/segmentation and sexual differentiation in ST. Moreover, many of the identified stage-specific proteins are useful as cestode developmental markers, and are potential targets for development of novel diagnostic methods and therapeutic drugs for cestodiases.


Assuntos
Larva/metabolismo , Estágios do Ciclo de Vida , Proteômica/métodos , Animais , Cestoides/química , Infecções por Cestoides/diagnóstico , Infecções por Cestoides/tratamento farmacológico , Proteínas de Helminto/análise , Proteínas de Helminto/fisiologia , Humanos , Mesocestoides/química , Reprodução Assexuada , Diferenciação Sexual
20.
Methods Mol Biol ; 1722: 3-20, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29264795

RESUMO

The bacterial surfaceome, comprising outer membrane-sorted and/or associated (i.e., cell transporters), cell surface-exposed (i.e., adhesins) and extracellularly secreted proteins (i.e., toxins), has been characterized in bacterial pathogens, such as Bordetella pertussis (Bp) to provide information for use in development of diagnostic and prevention strategies. This protein subset has clinical significance, as these bacterial proteins are often associated with attachment to host cells, microbial pathogenesis and antibody-mediated immunity. Here we describe classical surface membrane protein enrichment techniques, followed by proteomic methodologies, such as gel-free protein separation and antibody-affinity capture technologies in combination with nano-liquid chromatography mass spectrometry, for the identification and characterization of Bp surfaceome proteins.


Assuntos
Antígenos de Bactérias/análise , Proteínas da Membrana Bacteriana Externa/análise , Bordetella pertussis/isolamento & purificação , Proteômica/métodos , Sequência de Aminoácidos , Afinidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Bordetella pertussis/imunologia , Tampões (Química) , Carbonatos/química , Cromatografia Líquida , Bases de Dados de Proteínas , Imunoprecipitação/métodos , Espectrometria de Massas em Tandem
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