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1.
J Dairy Sci ; 105(10): 8497-8508, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35965128

RESUMO

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.


Assuntos
Insulinas , Leite , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Antioxidantes/metabolismo , Carnitina/análogos & derivados , Carnitina/análise , Bovinos , Colina/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Glutationa/metabolismo , Humanos , Lactação , Lipídeos/análise , Metaboloma , Leite/química , Nitrogênio/metabolismo , Fosforilcolina/análise , Fosforilcolina/metabolismo , Fosforilcolina/farmacologia , Ureia/metabolismo
2.
PLoS Negl Trop Dis ; 15(10): e0009819, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34606502

RESUMO

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is one of nineteen neglected tropical diseases. CD is a vector-borne disease transmitted by triatomines, but CD can also be transmitted through blood transfusions, organ transplants, T. cruzi-contaminated food and drinks, and congenital transmission. While endemic to the Americas, T. cruzi infects 7-8 million people worldwide and can induce severe cardiac symptoms including apical aneurysms, thromboembolisms and arrhythmias during the chronic stage of CD. However, these cardiac clinical manifestations and CD pathogenesis are not fully understood. Using spatial metabolomics (chemical cartography), we sought to understand the localized impact of chronic CD on the cardiac metabolome of mice infected with two divergent T. cruzi strains. Our data showed chemical differences in localized cardiac regions upon chronic T. cruzi infection, indicating that parasite infection changes the host metabolome at specific sites in chronic CD. These sites were distinct from the sites of highest parasite burden. In addition, we identified acylcarnitines and glycerophosphocholines as discriminatory chemical families within each heart region, comparing infected and uninfected samples. Overall, our study indicated global and positional metabolic differences common to infection with different T. cruzi strains and identified select infection-modulated pathways. These results provide further insight into CD pathogenesis and demonstrate the advantage of a systematic spatial perspective to understand infectious disease tropism.


Assuntos
Cardiomiopatia Chagásica/metabolismo , Miocárdio/metabolismo , Animais , Carnitina/análogos & derivados , Carnitina/análise , Carnitina/metabolismo , Cardiomiopatia Chagásica/parasitologia , Doença Crônica , Coração/parasitologia , Humanos , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos C3H , Miocárdio/química , Fosforilcolina/análise , Fosforilcolina/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/fisiologia
3.
J Dairy Res ; 88(3): 261-264, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34372949

RESUMO

In this work we report a lipidomics approach to study the effects of two diet systems on the composition of ovine milk. Milk from two groups of Sarda sheep grazing on 40% (P40) and 60% (P60) of pasture were analyzed by a UHPLC-QTOF-MS analytical platform and data submitted to multivariate statistical analysis. Pairwise partial least square discriminant analysis of the lipid profile of the data was carried out to classify samples and to find discriminant lipids. The two dietary groups were characterized by differences in triacylglycerols, phosphocholines and phosphatidylethanolamines levels. Discriminants of the P40 group were TG and PC containing in their backbone saturated medium chain FA thus suggesting greater de novo fatty synthesis in the mammary gland. On the other hand, the P60 group was characterized by TG and PC formed by unsaturated long chain FA originating from the diet or from lipid mobilization.


Assuntos
Dieta/veterinária , Lipidômica/métodos , Lipídeos/análise , Leite/química , Ovinos/metabolismo , Ração Animal , Animais , Feminino , Fosfatidiletanolaminas/análise , Fosforilcolina/análise , Triglicerídeos
4.
PLoS One ; 16(3): e0248632, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33735267

RESUMO

Abnormal cell membrane metabolism is associated with many neuropsychiatric disorders. Free phosphomonoesters and phosphodiesters, which can be detected by in vivo 31P magnetic resonance spectroscopy (MRS), are important cell membrane building blocks. However, the quantification of phosphoesters has been highly controversial even in healthy individuals due to overlapping signals from macromolecule membrane phospholipids (MP). In this study, high signal-to-noise ratio (SNR) cerebral 31P MRS spectra were acquired from healthy volunteers at both 3 and 7 Tesla. Our results indicated that, with minimal spectral interference from MP, the [phosphocreatine (PCr)]/[phosphocholine (PC) + glycerophosphocholine (GPC)] ratio measured at 7 Tesla agreed with its value expected from biochemical constraints. In contrast, the 3 Tesla [PCr]/[PC+GPC] ratio obtained using standard spectral fitting procedures was markedly smaller than the 7 Tesla ratio and than the expected value. The analysis suggests that the commonly used spectral model for MP may fail to capture its complex spectral features at 3 Tesla, and that additional prior knowledge is necessary to reliably quantify the phosphoester signals at low magnetic field strengths when spectral overlapping is significant.


Assuntos
Mapeamento Encefálico/métodos , Encéfalo/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Adolescente , Adulto , Idoso , Mapeamento Encefálico/instrumentação , Estudos de Viabilidade , Voluntários Saudáveis , Humanos , Imageamento por Ressonância Magnética/instrumentação , Masculino , Pessoa de Meia-Idade , Ressonância Magnética Nuclear Biomolecular/instrumentação , Fosfocreatina/análise , Fósforo/administração & dosagem , Fosforilcolina/análise , Adulto Jovem
5.
Chem Commun (Camb) ; 56(4): 539-542, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31829317

RESUMO

The first example of supramolecular recognition of phosphocholine by a cavitand receptor has been reported here. The chemical structure of the receptor has been optimized by DFT calculations. The recognition mechanism is based on a "multi-topic approach", which leads to highly efficient (K value up to 107 M-1), selective and sensitive (ppb level) sensing of phosphocholine. The recognition mechanism proposed here is similar to those exploited by Nature, and paves the way for the realization of new sensors with important applications in medicine and security fields.


Assuntos
Complexos de Coordenação/química , Fosforilcolina/análise , Zinco/química , Complexos de Coordenação/síntese química , Teoria da Densidade Funcional , Substâncias Macromoleculares/síntese química , Substâncias Macromoleculares/química , Estrutura Molecular
6.
Drug Test Anal ; 11(8): 1231-1237, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30950199

RESUMO

The erythrocyte membrane is composed of a phospholipid bilayer, which is known to undergo physicochemical changes during storage at low temperatures. This study was conducted to identify marker phospholipids that indicate alteration during deep-frozen storage and to determine the amount of marker phospholipids. Our research suggested a method to detect phospholipids by profiling analysis of thermally injured red blood cells (RBCs) without protecting agents. Human blood was stored at -80°C for 72 days. The RBC membrane phospholipids were extracted through a modified Bligh and Dyer method. Six selected phospholipids were analyzed and quantified using liquid chromatography-tandem mass spectrometry, and an in vitro model system was developed. The intracellular level of N-nervonoyl-D-erythro-sphingosylphosphorylcholine significantly increased in the thermally injured RBCs, and multiple biomarker candidates were evaluated by profiling analysis and mass spectrometry technology for targeted metabolomics.


Assuntos
Preservação de Sangue/métodos , Criopreservação/métodos , Eritrócitos/química , Fosfolipídeos/análise , Membrana Eritrocítica/química , Eritrócitos/citologia , Humanos , Fosforilcolina/análogos & derivados , Fosforilcolina/análise , Esfingosina/análogos & derivados , Esfingosina/análise
7.
Metabolomics ; 15(4): 55, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30927092

RESUMO

INTRODUCTION: Chronic exposure to high-glucose and free fatty acids (FFA) alone/or in combination; and the resulting gluco-, lipo- and glucolipo-toxic conditions, respectively, have been known to induce dysfunction and apoptosis of ß-cells in Diabetes. The molecular mechanisms and the development of biomarkers that can be used to predict similarities and differences behind these conditions would help in easier and earlier diagnosis of Diabetes. OBJECTIVES: This study aims to use metabolomics to gain insight into the mechanisms by which ß-cells respond to excess-nutrient stress and identify associated biomarkers. METHODS: INS-1E cells were cultured in high-glucose, palmitate alone/or in combination for 24 h to mimic gluco-, lipo- and glucolipo-toxic conditions, respectively. Biochemical and cellular experiments were performed to confirm the establishment of these conditions. To gain molecular insights, abundant metabolites were identified and quantified using 1H-NMR. RESULTS: No loss of cellular viability was observed in high-glucose while exposure to FFA alone/in combination with high-glucose was associated with increased ROS levels, membrane damage, lipid accumulation, and DNA double-strand breaks. Forty-nine abundant metabolites were identified and quantified using 1H-NMR. Chemometric pair-wise analysis in glucotoxic and lipotoxic conditions, when compared with glucolipotoxic conditions, revealed partial overlap in the dysregulated metabolites; however, the dysregulation was more significant under glucolipotoxic conditions. CONCLUSION: The current study compared gluco-, lipo- and glucolipotoxic conditions in parallel and elucidated differences in metabolic pathways that play major roles in Diabetes. o-phosphocholine and UDP-N-acetylglucosamine were identified as common dysregulated metabolites and their ratio was proposed as a potential biomarker for these conditions.


Assuntos
Células Secretoras de Insulina/metabolismo , Fosforilcolina/análise , Uridina Difosfato N-Acetilglicosamina/análise , Animais , Apoptose , Biomarcadores/sangue , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/metabolismo , Glucose/efeitos adversos , Glucose/metabolismo , Células Secretoras de Insulina/fisiologia , Palmitatos/efeitos adversos , Palmitatos/metabolismo , Fosforilcolina/sangue , Ratos , Uridina Difosfato N-Acetilglicosamina/sangue
8.
Food Chem ; 281: 251-260, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30658755

RESUMO

Lipid hydrolysis and oxidation occurred in Argopecten irradians adductor muscle during hot air drying. Using an in vivo imaging system, we found that antioxidants of bamboo leaves (AOB) could diffuse into the adductor muscle upon marinating. Both tea polyphenols (TP) and AOB efficiently retarded lipid oxidation but had a slight effect on lipid hydrolysis during drying process. The in situ antioxidant mechanisms of AOB as well as TP were revealed, including quenching of free radicals detected by electron spin resonance, chelating metal ions determined by confocal laser scanning microscopy and inhibiting lipoxygenase. Less than 8% of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in AOB and TP marinated adductor muscle were decreased compared to more than 28% decrease in control adductor muscle during the drying process. Overall, these natural antioxidants, TP and AOB, efficiently maintained high nutritive value of adductor muscle, especially, their lipid quality.


Assuntos
Antioxidantes/análise , Dessecação , Manipulação de Alimentos , Pectinidae , Polifenóis/análise , Alimentos Marinhos/análise , Chá/química , Animais , Ácidos Docosa-Hexaenoicos/análise , Ácido Eicosapentaenoico/análise , Ácidos Graxos Insaturados/análise , Metabolismo dos Lipídeos , Valor Nutritivo , Fosforilcolina/análise , Extratos Vegetais/análise , Folhas de Planta/química , Sasa/química , Substâncias Reativas com Ácido Tiobarbitúrico/análise
9.
Clin Neuroradiol ; 29(1): 27-36, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28983683

RESUMO

PURPOSE: Previous ex vivo spectroscopic data from tissue samples revealed differences in phospholipid metabolites between isocitrate dehydrogenase mutated (IDHmut) and IDH wildtype (IDHwt) gliomas. We investigated whether these changes can be found in vivo using 1H-decoupled 31P magnetic resonance spectroscopic imaging (MRSI) with 3D chemical shift imaging (CSI) at 3 T in patients with low and high-grade gliomas. METHODS: The study included 33 prospectively enrolled, mostly untreated patients who met spectral quality criteria according to the World Health Organization (WHO II n = 7, WHO III n = 17, WHO IV n = 9; 25 patients IDHmut, 8 patients IDHwt). The MRSI protocol included 1H decoupled 31P MRSI with 3D CSI (3D 31P CSI), 2D 1H CSI and a 1H single voxel spectroscopy sequence (TE 30 ms) from the tumor area. For 1H MRS, absolute metabolite concentration values were calculated (phantom replacement method). For 31P MRS, metabolite intensity ratios were calculated for the choline (C) and ethanolamine (E)-containing metabolites. RESULTS: In our patient cohort we did not find significant differences for the ratio of phosphocholine (PC) and phosphoethanolamine (PE), PC/PE, (p = 0.24) for IDHmut compared to IDHwt gliomas. Furthermore, we found no elevated ratios of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE), GPC/GPE, (p = 0.68) or GPC/PE (p = 0.12) for IDHmut gliomas. Even the ratio (PC+GPC)/(PE+GPE) showed no significant differences with respect to mutation status (p = 0.16). Nonetheless, changes related to tumor grade regarding intracellular pH (pHi) and phospholipid metabolism as well as absolute metabolite concentrations of co-registered 2D 1H CSI data for tumor and control tissue showed the anticipated results. CONCLUSION: Using 3D-CSI data acquisition, in vivo 31P MR spectroscopic measurement of phospholipid metabolites could not distinguish between IDHmut and IDHwt.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Isocitrato Desidrogenase/genética , Espectroscopia de Ressonância Magnética/métodos , Adulto , Idoso , Análise de Variância , Astrocitoma/enzimologia , Astrocitoma/genética , Astrocitoma/patologia , Astrocitoma/terapia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Diagnóstico Diferencial , Etanolaminas/análise , Etanolaminas/metabolismo , Feminino , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Glioma/genética , Glioma/patologia , Glioma/terapia , Glutaratos/análise , Glutaratos/metabolismo , Glicerilfosforilcolina/análise , Humanos , Hidrogênio , Isocitrato Desidrogenase/metabolismo , Isoenzimas/análise , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Oligodendroglioma/enzimologia , Oligodendroglioma/genética , Oligodendroglioma/patologia , Oligodendroglioma/terapia , Fosfatidiletanolaminas/análise , Fosfatidiletanolaminas/metabolismo , Isótopos de Fósforo , Fosforilcolina/análise , Fosforilcolina/metabolismo , Estudos Prospectivos , Carga Tumoral
10.
Med Microbiol Immunol ; 207(5-6): 329-338, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30056510

RESUMO

Phosphorylcholine (ChoP) is covalently incorporated into bacterial surface structures, contributing to host mimicry and promoting adhesion to surfaces. Our aims were to determine the frequency of ChoP display among Aggregatibacter actinomycetemcomitans strains, to clarify which surface structures bear ChoP, and whether ChoP-positivity relates to serum killing. The tested oral (N = 67) and blood isolates (N = 27) represented 6 serotypes. Mab TEPC-15 was used for immunoblotting of cell lysates and fractions and for immunofluorescence microscopy of cell surface-bound ChoP. The lysates were denatured with urea for hidden ChoP or treated with proteinase K to test whether it binds to a protein. Three ChoP-positive and two ChoP-negative strains were subjected to serum killing in the presence/absence of CRP and using Ig-depleted serum as complement source. Cell lysates and the first soluble cellular fraction revealed a < 10 kDa band in immunoblots. Among 94 strains, 27 were ChoP positive. No difference was found in the prevalence of ChoP-positive oral (21/67) and blood (6/27) strains. Immunofluorescence microscopy corresponded to the immunoblot results. Proteinase K abolished ChoP reactivity, whereas urea did not change the negative result. The TEPC-15-reactive protein was undetectable in Δflp1 mutant strain. The survival rate of serotype-b strains in serum was 100% irrespective of ChoP, but that of serotype-a was higher in ChoP-positive (85%) than ChoP-negative (71%) strains. The results suggest that a third of rough-colony strains harbor ChoP and that ChoP is attached to fimbrial subunit protein Flp1. It further seems that ChoP-positivity does not enhance but may reduce A. actinomycetemcomitans susceptibility to serum killing.


Assuntos
Aggregatibacter actinomycetemcomitans/química , Aggregatibacter actinomycetemcomitans/imunologia , Proteínas de Bactérias/química , Fosforilcolina/análise , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Proteínas de Bactérias/genética , Sangue/microbiologia , Atividade Bactericida do Sangue , Deleção de Genes , Humanos , Immunoblotting , Viabilidade Microbiana , Microscopia de Fluorescência , Boca/microbiologia , Infecções por Pasteurellaceae/microbiologia , Sorogrupo
11.
Talanta ; 178: 538-544, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29136859

RESUMO

The chemical shifts of several important endogenous phosphorus compounds under different pH conditions were explored, including adenosine-5'-triphosphate, adenosine-5'-diphosphate, adenosine-5'-monophosphate, phosphorylcholine and phosphorylethanolamine. Their 31P NMR and 1H NMR chemical shifts were all pH-sensitive in the similar pH range. Two dimensional (2D) 1H-31P NMR spectra were found helpful to identify these endogenous phosphorus markers in biological samples from rather complicated NMR spectra. Herein, for the first time, a pH sensor based on 2D 1H-31P NMR was established and applied to biological samples analysis with pH values determined in good agreement with those by potentiometric method. Apart from being simple, green, rapid and less sample-consuming, information concerning both the endogenous phosphorus markers and pH status could be attained in a single NMR run, which demonstrated the great potential of this method in rare sample analysis and even disease diagnosis.


Assuntos
Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Difosfato de Adenosina/análise , Difosfato de Adenosina/urina , Monofosfato de Adenosina/análise , Monofosfato de Adenosina/urina , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/urina , Etanolaminas/análise , Etanolaminas/urina , Sucos de Frutas e Vegetais/análise , Células Hep G2 , Humanos , Malus , Fosforilcolina/análise , Fosforilcolina/urina
12.
J Am Soc Mass Spectrom ; 28(8): 1729-1732, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28508286

RESUMO

Corn seed tissue sections were prepared by the tape support method using an adhesive tape, and mass spectrometry imaging (MSI) was performed. The effect of heat generated during sample preparation was investigated by time-of-flight secondary mass spectrometry (TOF-SIMS) imaging of corn seed tissue prepared by the tape support and the thaw-mounted methods. Unlike thaw-mounted sample preparation, the tape support method does not cause imaging distortion because of the absence of heat, which can cause migration of the analytes on the sample. By applying the tape-support method, the corn seed tissue was prepared without structural damage and MSI with accurate spatial information of analytes was successfully performed. Graphical Abstract ᅟ.


Assuntos
Sementes/química , Espectrometria de Massa de Íon Secundário/métodos , Zea mays/química , Colina/análise , Desenho de Equipamento , Ácido Palmítico/análise , Fosforilcolina/análise , Sementes/embriologia , Espectrometria de Massa de Íon Secundário/instrumentação , Zea mays/embriologia
13.
J Am Soc Mass Spectrom ; 28(8): 1552-1561, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28462493

RESUMO

Maturation of metabolomics has brought a deeper appreciation for the importance of isomeric identity of lipids to their biological role, mirroring that for proteoforms in proteomics. However, full characterization of the lipid isomerism has been thwarted by paucity of rapid and effective analytical tools. A novel approach is ion mobility spectrometry (IMS) and particularly differential or field asymmetric waveform IMS (FAIMS) at high electric fields, which is more orthogonal to mass spectrometry. Here we broadly explore the power of FAIMS to separate lipid isomers, and find a ~75% success rate across the four major types of glycero- and phospho- lipids (sn, chain length, double bond position, and cis/trans). The resolved isomers were identified using standards, and (for the first two types) tandem mass spectrometry. These results demonstrate the general merit of incorporating high-resolution FAIMS into lipidomic analyses. Graphical Abstract ᅟ.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Lipídeos/análise , Espectrometria de Massas em Tandem/métodos , Glicerídeos/análise , Íons/análise , Isomerismo , Fosforilcolina/análise , Espectrometria de Massas por Ionização por Electrospray/métodos
14.
J Pharm Biomed Anal ; 135: 160-166, 2017 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-28033553

RESUMO

Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly endemic in resource-poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We developed and validated an LC-MS/MS method for the quantification of miltefosine with this novel sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within ±10.8%), precision (CV%≤11.9%), recovery, carry-over and matrix effect. VAMS samples were stable for at least one month at room temperature and 37°C. The impact of haematocrit on assay accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A clinical validation will be required to investigate whether VAMS is an appropriate and cost-effective alternative sampling method to conventional DBS sampling.


Assuntos
Antiprotozoários/sangue , Teste em Amostras de Sangue Seco/métodos , Fosforilcolina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Antiprotozoários/análise , Cromatografia Líquida/métodos , Humanos , Fosforilcolina/análise , Fosforilcolina/sangue , Manejo de Espécimes/métodos
15.
PLoS Negl Trop Dis ; 10(12): e0005166, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27906979

RESUMO

BACKGROUND: The pig parasite Ascaris suum plays and important role in veterinary medicine and represents a suitable model for A. lumbricoides, which infects over 800 million people. In pigs, continued exposure to Ascaris induces immunity at the level of the gut, protecting the host against migrating larvae. The objective of this study was to identify and characterize parasite antigens targeted by this local immune response that may be crucial for parasite invasion and establishment and to evaluate their protective and diagnostic potential. METHODOLOGY/PRINCIPAL FINDINGS: Pigs were immunized by trickle infection for 30 weeks, challenged with 2,000 eggs at week 32 and euthanized two weeks after challenge. At necropsy, there was a 100% reduction in worms recovered from the intestine and a 97.2% reduction in liver white spots in comparison with challenged non-immune control animals. Antibodies purified from the intestinal mucus or from the supernatant of cultured antibody secreting cells from mesenteric lymph nodes of immune pigs were used to probe L3 extracts to identify antibody targets. This resulted in the recognition of a 12kDa antigen (As12) that is actively shed from infective Ascaris L3. As12 was characterized as a phosphorylcholine-containing glycolipid-like antigen that is highly resistant to different enzymatic and chemical treatments. Vaccinating pigs with an As12 fraction did not induce protective immunity to challenge infection. However, serological analysis using sera or plasma from experimentally infected pigs or naturally infected humans demonstrated that the As12 ELISA was able to detect long-term exposure to Ascaris with a high diagnostic sensitivity (98.4% and 92%, respectively) and specificity (95.5% and 90.0%) in pigs and humans, respectively. CONCLUSIONS/SIGNIFICANCE: These findings show the presence of a highly stage specific, glycolipid-like component (As12) that is actively secreted by infectious Ascaris larvae and which acts as a major antibody target in infected humans and pigs.


Assuntos
Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Ascaríase/imunologia , Ascaríase/veterinária , Ascaris suum/imunologia , Glicolipídeos/imunologia , Fosforilcolina/imunologia , Doenças dos Suínos/imunologia , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Ascaríase/parasitologia , Ascaris suum/química , Ascaris suum/genética , Feminino , Glicolipídeos/análise , Humanos , Larva/química , Larva/genética , Larva/imunologia , Masculino , Fosforilcolina/análise , Suínos , Doenças dos Suínos/parasitologia
16.
Infect Immun ; 84(3): 765-74, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26729761

RESUMO

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.


Assuntos
Infecções por Haemophilus/microbiologia , Haemophilus influenzae/metabolismo , Haemophilus/metabolismo , Lipopolissacarídeos/química , Ácido N-Acetilneuramínico/análise , Fosforilcolina/análise , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Haemophilus/química , Haemophilus/classificação , Haemophilus/isolamento & purificação , Haemophilus influenzae/química , Haemophilus influenzae/classificação , Haemophilus influenzae/isolamento & purificação , Humanos , Lipopolissacarídeos/metabolismo , Espectrometria de Massas , Ácido N-Acetilneuramínico/metabolismo , Fosforilcolina/metabolismo
17.
J Biol Regul Homeost Agents ; 29(2): 443-50, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26122235

RESUMO

This study discusses the application of magnetic resonance spectrum (MRS) to evaluate the efficacy of antiviral therapy in the treatment of liver cirrhosis caused by chronic hepatitis C and hepatitis C, based on metabolite detection. A total of 54 patients with liver cirrhosis caused by chronic hepatitis C and hepatitis C were selected and divided into treatment group and control group. 31P-MRS imaging was carried out on patients in the two groups both before receiving antiviral treatment and 6 months after treatment to compare the change of metabolite ratio (PE+PC)/(GPE+GPC). It was revealed that no statistically significant difference was found in the comparison of (PC+PE)/(GPC+GPE) ratio in the two groups before treatment, but the difference was found 6 months after treatment; ratio of (PC+PE)/ (GPC+GPE) in the treatment group distinctly decreased 6 months after treatment compared to before treatment, with a statistically significant difference, while the control group had no remarkable change or statistical significance. Moreover, 32 patients were found with sustained virus response to antiviral therapy. Of these, 25 patients possessed a decreased ratio of (PC+PE)/ (GPC+GPE), 4 remained without change and 3 had a slightly increased ratio after antiviral treatment. Of 12 patients with no response, 1 had a decreased ratio of (PC+PE)/ (GPC+GPE), 2 remained without change and 9 had a slightly increased ratio. The differences were all statistically significant in comparison of the two groups. 31P-MRS is thought to be effective for evaluating the efficacy of antiviral therapy through non-invasive detection of liver energy metabolism.


Assuntos
Trifosfato de Adenosina/análise , Antivirais/uso terapêutico , Monitoramento de Medicamentos/métodos , Etanolaminas/análise , Glicerilfosforilcolina/análise , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Fígado/química , Espectroscopia de Ressonância Magnética , Fosfatidiletanolaminas/análise , Fosforilcolina/análise , Polietilenoglicóis/uso terapêutico , Ribavirina/uso terapêutico , Adulto , Idoso , Feminino , Hepacivirus/isolamento & purificação , Hepatite C/metabolismo , Hepatite C Crônica/complicações , Hepatite C Crônica/metabolismo , Humanos , Cirrose Hepática/etiologia , Cirrose Hepática/metabolismo , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Isótopos de Fósforo , RNA Viral/sangue , Proteínas Recombinantes/uso terapêutico
18.
Artigo em Inglês | MEDLINE | ID: mdl-26160472

RESUMO

Phagocytes, the physiological compartment in which Leishmania parasites reside, are the main site of action of the drug miltefosine, but the intracellular pharmacokinetics of miltefosine remain unexplored. We developed a bioanalytical method to quantify miltefosine in human peripheral blood mononuclear cells (PBMCs), expanding from an existing high performance liquid chromatography-tandem mass spectrometry method for the quantification of miltefosine in plasma. The method introduced deuterated miltefosine as an internal standard. Miltefosine was extracted from PBMC pellets by addition of 62.5% methanol. Supernatant was collected, evaporated and reconstituted in plasma. Chromatographic separation was performed on a reversed phase C18 column and detection with a triple-quadrupole mass spectrometer. Miltefosine was quantified using plasma calibration standards ranging from 4 to 1000ng/mL. This method was validated with respect to its PBMC matrix effect, selectivity, recovery and stability. No matrix effect could be observed from the PBMC content (ranging from 0.17 to 26.3×10(6)PBMCs) reconstituted in plasma, as quality control samples were within 3.0% of the nominal concentration (precision less than 7.7%). At the lower limit of quantitation of 4 ng/mL plasma, corresponding to 0.12ng/10(6) PBMCs in a typical clinical sample, measured concentrations were within 8.6% of the nominal value. Recovery showed to be reproducible as adding additional pre-treatment steps did not increase the recovery with more than 9%. This method was successfully applied to measure intracellular miltefosine concentrations in PBMC samples from six cutaneous leishmaniasis patients up to one month post-treatment.


Assuntos
Antiprotozoários/análise , Cromatografia Líquida de Alta Pressão/métodos , Leucócitos Mononucleares/química , Fosforilcolina/análogos & derivados , Espectrometria de Massas em Tandem/métodos , Humanos , Fosforilcolina/análise
19.
Rev. bras. anestesiol ; 65(3): 180-185, May-Jun/2015. tab
Artigo em Inglês | LILACS | ID: lil-748916

RESUMO

INTRODUCTION: The rates of multiresistant bacteria colonization or infection (MRB+) development in intensive care units are very high. The aim of this study was to determine the possible association between the risk of development of nosocomial infections and increased daily nurse workload due to understaffing in intensive care unit. METHODS: We included 168 patients. Intensity of workload and applied procedures to patients were scored with the Project de Recherché en Nursing and the Omega scores, respectively. The criteria used for infections were those defined by the Centers for Disease Control. RESULTS: Of the 168 patients, 91 (54.2%) were female and 77 (45.8%) were male patients. The mean age of female and male was 64.9 ± 6.2 years and 63.1 ± 11.9 years, respectively. The mean duration of hospitalization in intensive care unit was 18.4 ± 6.1 days. Multiresistant bacteria were isolated from cultures of 39 (23.2%) patients. The development of MRB+ infection was correlated with length of stay, Omega 1, Omega 2, Omega 3, Total Omega, daily PRN, and Total PRN (p < 0.05). There was no correlation between development of MRB+ infection with gender, age and APACHE-II scores (p > 0.05). CONCLUSION: The risk of nosocomial infection development in an intensive care unit is directly correlated with increased nurse workload, applied intervention, and length of stay. Understaffing in the intensive care unit is an important health problem that especially affects care-needing patients. Nosocomial infection development has laid a heavy burden on the economy of many countries. To control nosocomial infection development in the intensive care unit, nurse workload, staffing level, and working conditions must be arranged. .


INTRODUÇÃO: As taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes (BMR+) em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem devido à insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: Cento e sessenta e oito pacientes foram incluídos. O volume da carga de trabalho e os procedimentos realizados em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os definidos pelos Centros de Controle de Doenças. RESULTADOS: Dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do sexo masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR+ foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR+ e gênero, idade e escores no APACHE-II (p > 0,05). CONCLUSÃO: O risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga ...


INTRODUÇÃO: as taxas de desenvolvimento de infecção ou colonização por bactérias multirresistentes [BMR (+)] em unidades de terapia intensiva são muito elevadas. O objetivo deste estudo foi determinar a possível associação entre o risco de desenvolvimento de infecções hospitalares e o aumento da carga de trabalho diária da equipe de enfermagem por causa da insuficiência de pessoal em unidade de terapia intensiva. MÉTODOS: foram incluídos 168 pacientes. O volume da carga de trabalho e os procedimentos feitos em pacientes foram avaliados com o uso de instrumentos de medidas como o Projeto de Pesquisa em Enfermagem (Project de Recherché en Nursing) e o Omega, respectivamente. Os critérios usados para definir infecções foram os estabelecidos pelos Centros de Controle de Doenças. RESULTADOS: dos 168 pacientes, 91 (54,2%) eram do sexo feminino e 77 (45,8%) do masculino. As médias das idades de mulheres e homens foram 64,9 ± 6,2 e 63,1 ± 11,9 anos, respectivamente. A média do tempo de internação em unidade de terapia intensiva foi de 18,4 ± 6,1 dias. As bactérias multirresistentes foram isoladas a partir de culturas de 39 (23,2%) pacientes. O desenvolvimento de infecção por BMR (+) foi correlacionado com tempo de internação, Omega 1, Omega 2, Omega 3, Omega total, PPE diário e PPE total (p < 0,05). Não houve correlação entre desenvolvimento de infecção por BMR (+) e gênero, idade e escores no Apache-II (p > 0,05). CONCLUSÃO: o risco de desenvolvimento de infecção hospitalar em unidade de terapia intensiva está diretamente relacionado com o aumento da carga de trabalho de enfermagem, as intervenções praticadas e o tempo de internação. A falta de pessoal em unidade de terapia intensiva é um problema de saúde importante que afeta principalmente os pacientes que requerem cuidados. A infecção hospitalar colocou um fardo pesado sobre a economia de muitos países. Para controlar o desenvolvimento de infecção hospitalar em UTI, a carga de trabalho ...


INTRODUCCIÓN: Las tasas de desarrollo de infección o colonización por bacterias multirresistentes en unidades de cuidados intensivos son muy elevadas. El objetivo de este estudio fue determinar la posible asociación entre el riesgo de desarrollo de infecciones hospitalarias y el aumento de la carga de trabajo diaria del equipo de enfermería debido a la falta de personal en la unidad de cuidados intensivos. MÉTODOS: Ciento sesenta y ocho pacientes fueron incluidos. El volumen de la carga de trabajo y los procedimientos realizados en pacientes fueron evaluados con el uso de instrumentos de medidas como el Proyecto de Investigación en Enfermería (Project de Recherché en Nursing) y el Omega, respectivamente. Los criterios usados para definir infecciones fueron los definidos por los Centros de Control de Enfermedades. RESULTADOS: De los 168 pacientes, 91 (54,2%) eran del sexo femenino y 77 (45,8%) del sexo masculino. La edad media de las mujeres y de los hombres fueron 64,9 ± 6,2 y 63,1 ± 11,9 años, respectivamente. El tiempo medio de ingreso en la unidad de cuidados intensivos fue de 18,4 ± 6,1 días. Las bacterias multirresistentes fueron aisladas a partir de cultivos de 39 (23,2%) pacientes. El desarrollo de infección por bacterias multirresistentes fue correlacionado con el tiempo de ingreso, Omega 1, Omega 2, Omega 3, Omega total, PPE diario y PPE total (p < 0,05). No hubo correlación entre el desarrollo de la infección por bacterias multirresistentes y el sexo, la edad y las puntuaciones en el APACHE-II (p > 0,05). CONCLUSIÓN: El riesgo de desarrollo de infección hospitalaria en una unidad de cuidados intensivos está directamente relacionado con el aumento de la carga de trabajo de enfermería, las intervenciones practicadas y el tiempo de ingreso. La falta de personal en la unidad de cuidados intensivos es un problema de sanidad importante que afecta principalmente a los pacientes que necesitan esos cuidados. La infección hospitalaria ...


Assuntos
Criança , Feminino , Humanos , Masculino , Núcleos Cerebelares/patologia , Transtornos do Espectro Alcoólico Fetal/patologia , Ácido Aspártico/análise , Ácido Aspártico/análogos & derivados , Encéfalo/patologia , Estudos de Casos e Controles , Núcleos Cerebelares/química , Glicerilfosforilcolina/análise , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Neuroimagem , Fosforilcolina/análise
20.
Int J Oral Sci ; 7(2): 103-9, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-25655010

RESUMO

Secondary caries due to biofilm acids is a primary cause of dental composite restoration failure. To date, there have been no reports of dental composites that can repel protein adsorption and inhibit bacteria attachment. The objectives of this study were to develop a protein-repellent dental composite by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) and to investigate for the first time the effects of MPC mass fraction on protein adsorption, bacteria attachment, biofilm growth, and mechanical properties. Composites were synthesized with 0 (control), 0.75%, 1.5%, 2.25%, 3%, 4.5% and 6% of MPC by mass. A commercial composite was also tested as a control. Mechanical properties were measured in three-point flexure. Protein adsorption onto the composite was determined by the microbicinchoninic acid method. A human saliva microcosm biofilm model was used. Early attachment at 4 h, biofilm at 2 days, live/dead staining and colony-forming units (CFUs) of biofilms grown on the composites were investigated. Composites with MPC of up to 3% had mechanical properties similar to those without MPC and those of the commercial control, whereas 4.5% and 6% MPC decreased the mechanical properties (P<0.05). Increasing MPC from 0 to 3% reduced the protein adsorption on composites (P<0.05). The composite with 3% MPC had protein adsorption that was 1/12 that of the control (P<0.05). Oral bacteria early attachment and biofilm growth were also greatly reduced on the composite with 3% MPC, compared to the control (P<0.05). In conclusion, incorporation of MPC into composites at 3% greatly reduced protein adsorption, bacteria attachment and biofilm CFUs, without compromising mechanical properties. Protein-repellent composites could help to repel bacteria attachment and plaque build-up to reduce secondary caries. The protein-repellent method might be applicable to other dental materials.


Assuntos
Resinas Compostas/química , Metacrilatos/análise , Fosforilcolina/análogos & derivados , Proteínas/química , Adsorção , Biofilmes , Contagem de Colônia Microbiana , Placa Dentária/microbiologia , Fosforilcolina/análise
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