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1.
Mol Immunol ; 132: 60-78, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545626

RESUMO

Toll-like receptors (TLRs) play a crucial role in the innate immune system, which is the first line of defence against pathogens and pathogenic products in fish. In the present study, we cloned the full-length cDNA and genome sequences of two TLR13 s (OnTLR13a, OnTLR13b) from Nile tilapia (Oreochromis niloticus). TLR family motifs, i.e., the leucine-rich repeat (LRR) domains and Toll/interleukin (IL)-1 receptor (TIR) domains, were conserved in the putative proteins OnTLR13a and OnTLR13b, with fifteen LRR domains and one TIR domain. Four exons and three introns were identified in the OnTLR13a genome sequence, and three exons and two introns were identified in the OnTLR13b genome sequence. In healthy Nile tilapia tissues, OnTLR13a and OnTLR13b were ubiquitously expressed in all 11 tested tissues/organs. The highest expression levels were observed in the spleen (OnTLR13a) and blood (OnTLR13b), and the lowest expression levels were observed in the liver (OnTLR13a) and stomach (OnTLR13b). The expression level of OnTLR13b at 5.5 days postfertilization (dpf) was significantly higher than that at the other 8 time points (2.5, 3.5, 4.5, 5, 6, 6.5, 7.5 and 8.5 dpf). Upon stimulation with an intraperitoneal injection of 200 µL (107 CFU/mL) Streptococcus agalactiae, the expression levels of OnTLR13a and OnTLR13b were significantly upregulated in the intestine and gill. After cotransfection with MyD88, OnTLR13a significantly increased MyD88-dependent NF-κB activation in 293 T cells. However, OnTLR13b significantly impaired MyD88-dependent NF-κB activation. In addition, TLR13a slightly increased MyD88-dependent AP-1 activation, and TLR13b significantly increased MyD88-dependent AP-1 activation. TLR13a significantly increased MyD88-dependent interferon-ß (IFN-ß) activation, and TLR13b had no effect on MyD88-dependent IFN-ß activation. These findings suggest that although the deduced protein structure of OnTLR13 is evolutionarily conserved between OnTLR13 and other TLR members, its signal transduction function is markedly different. Co-immunoprecipitation (Co-IP) assays showed that both OnTLR13a and OnTLR13b could interact with OnMyD88. RNA pulldown assays showed that TLR13a and TLR13b could combine with the 23S rRNA of S. agalactiae. These results indicate that TLR13a and TLR13b play important roles in the innate immune response against bacterial infection in Nile tilapia.


Assuntos
Ciclídeos/genética , Ciclídeos/imunologia , Imunidade Inata/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Streptococcus agalactiae/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Sangue/metabolismo , Ciclídeos/metabolismo , Ciclídeos/microbiologia , Éxons , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Interferon beta/metabolismo , Íntrons , Fígado/metabolismo , NF-kappa B/metabolismo , Filogenia , Domínios Proteicos , RNA Ribossômico 23S/genética , Alinhamento de Sequência , Transdução de Sinais/imunologia , Baço/metabolismo , Fator de Transcrição AP-1/metabolismo
2.
Molecules ; 26(3)2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513993

RESUMO

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.


Assuntos
Amicacina/química , Sangue/metabolismo , Plasma/química , Acetonitrilos/química , Hidróxido de Amônia/química , Animais , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Feminino , Camundongos
3.
Methods Mol Biol ; 2241: 15-25, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486724

RESUMO

The choice of isolation technique for human peripheral blood eosinophils contributes to the understanding of clinically relevant data derived from in vitro research. Since the 1990s, eosinophils have been conventionally isolated via density gradient centrifugation followed by negative immunomagnetic selection using anti-CD16 antibody-coated magnetic beads. Due to recent advancements in molecular techniques, "newer" methods have been made commercially available that drastically reduce user handling and processing time while maintaining high population purity. Here, we describe an isolation procedure using one of these methods, the human MACSxpress® Whole Blood Isolation Kit, as well as outline protocols for differential staining and flow cytometry analysis to evaluate the purity and activation state of isolated cells. In addition, we highlight an in vitro degranulation assay that may be used to verify the intact functionality of the isolated eosinophils.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Eosinófilos/fisiologia , Sangue/metabolismo , Células Sanguíneas/citologia , Centrifugação com Gradiente de Concentração/métodos , Eosinófilos/metabolismo , Citometria de Fluxo/métodos , Humanos , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Receptores de IgG/imunologia
4.
Methods Mol Biol ; 2241: 27-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486725

RESUMO

Eosinophils are granulocytes involved mainly in allergic inflammation and parasitic responses and constitute 1-5% of the circulating leukocytes in human healthy subjects. New immunotherapies targeting eosinophils have been developed and evaluated recently, and the availability of animal models that could mimic human eosinophil responses is important to consider. Differences in eosinophil biology and pathogenesis between humans and murine models have limited their utility in some settings. Isolation of viable eosinophils from rhesus macaque blood suitable for ex vivo and in vitro experimentation could provide a valuable tool for the study of eosinophil-targeted therapies and for the exploration of eosinophilic associated responses. Here, a new technique for the isolation of human eosinophils from rhesus macaque blood by negative selection from whole blood is described.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Eosinófilos/fisiologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Centrifugação com Gradiente de Concentração/métodos , Eosinófilos/metabolismo , Citometria de Fluxo/métodos , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Macaca mulatta/imunologia , Receptores de IgG/imunologia
5.
Methods Mol Biol ; 2241: 49-58, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33486727

RESUMO

Flow cytometry is a critical tool that can be employed to detect unique cells and to isolate cells from tissues based on their antigen profiles. While mouse eosinophils can be readily detected by one or more distinct antigen profiles, many of these strategies do not result in accurate eosinophil counts. We present here our basic protocol, which permits quantitative detection of eosinophils and isolation of eosinophils from bone marrow, spleen, and lung tissue of allergen-challenged wild-type and unchallenged IL5 transgenic mice. With small protocol variations, eosinophils can be isolated from small intestines and muscle tissue, the latter from infiltrates characteristic of muscular dystrophy (mdx) mice.


Assuntos
Separação Celular/métodos , Eosinófilos/citologia , Citometria de Fluxo/métodos , Alérgenos/imunologia , Animais , Sangue/metabolismo , Células Sanguíneas/citologia , Medula Óssea/imunologia , Células da Medula Óssea/citologia , Eosinófilos/metabolismo , Eosinófilos/fisiologia , Feminino , Separação Imunomagnética/métodos , Contagem de Leucócitos/métodos , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Receptores de IgG/imunologia , Baço/citologia , Baço/imunologia
6.
Int J Nanomedicine ; 16: 443-456, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33505159

RESUMO

Introduction: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort. Methods: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement. Results: More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found. Conclusion: We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.


Assuntos
Atletas , Sangue/metabolismo , Nanopartículas/química , Adulto , Contagem de Células Sanguíneas , Proteína C-Reativa/análise , Eritrócitos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Interleucina-6/sangue , Lipídeos/química , Masculino , Pessoa de Meia-Idade , Esforço Físico/fisiologia , Fator de Necrose Tumoral alfa/sangue , Adulto Jovem
7.
J Chromatogr A ; 1632: 461587, 2020 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-33059177

RESUMO

Supercritical carbon dioxide (scCO2), the main fluid in the mobile phase for supercritical fluid chromatography (SFC), is non-polar. The majority of polar compounds are little soluble in scCO2, thereby rendering them poor candidates for achieving separation by carbon dioxide-based SFC. There is no reported method for the comprehensive analysis of hydrophilic metabolites by SFC with mobile phases comprising a high CO2 ratio. In this study, we investigated the effect of additives in the modifier for enabling the application of SFC to profile diverse polar compounds for metabolomics. Eleven types of columns were screened by using proteinogenic amino acids as the model compounds. The addition of water and acids (formic acid and trifluoroacetic acid (TFA)) to the modifier was also investigated to improve the solubility of the polar compounds and mitigate the unfavorable interaction between the stationary phase and the polar compounds. A significant improvement in the peak shapes of the amino acids was observed upon addition of TFA. The CO2/modifier ratio and TFA concentration in the mobile phases were investigated using the CROWNPAK CR-I (+) column, which showed the best performance during the column-screening. The CO2/methanol/water/TFA ratio of 70/27/3/0.15 (v/v/v/v) was determined as the optimized mobile phase composition. Furthermore, the applicability of the optimized analytical method to other polar compounds was examined; 100 cationic and amphoteric compounds with predicted logPow values that ranged from -5.9 to 1.7 could be simultaneously analyzed without derivatization. Anionic compounds such as organic acids, phosphates, and sugars were excluded from the target analytes. Most of the previously reported SFC methods for analyzing polar compounds employ a gradient elution and require the use of high modifier ratios at 40% or more. In the proposed method, the use of water and TFA enabled the rapid and simultaneous analysis under isocratic elution within 10 min, even with a high CO2 ratio of 70%. Additionally, a rat serum extract was analyzed using the optimized conditions, and 43 polar metabolites were successfully detected. This result demonstrates the applicability of the SFC/tandem mass spectrometry method to real samples.


Assuntos
Cromatografia com Fluido Supercrítico/métodos , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/análise , Animais , Sangue/metabolismo , Dióxido de Carbono/química , Formiatos/química , Interações Hidrofóbicas e Hidrofílicas , Metanol/química , Ratos , Ácido Trifluoracético/química , Água/química
8.
PLoS One ; 15(9): e0230984, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32946446

RESUMO

Insecticide resistance genes are often associated with pleiotropic effects on various mosquito life-history traits. However, very little information is available on the impact of insecticide resistance on blood feeding process in mosquitoes. Here, using two recently detected DNA-based metabolic markers in the major malaria vector, An. funestus, we investigated how metabolic resistance genes could affect the blood meal intake. After allowing both the field F1 and lab F8 Anopheles funestus strains to feed on the human arm for 30 minutes, we assessed the association between key parameters of blood meal process including, probing time, feeding duration, blood feeding success, blood meal size, and markers of glutathione S-transferase (L119F-GSTe2) and cytochrome P450 (CYP6P9a_R)-mediated metabolic resistance. None of the parameters of blood meal process was associated with L119F-GSTe2 genotypes. By contrast, for CYP6P9a_R, homozygous resistant mosquitoes were significantly more able to blood-feed than homozygous susceptible (OR = 3.3; CI 95%: 1.4-7.7; P = 0.01) mosquitoes. Moreover, the volume of blood meal ingested by CYP6P9a-SS mosquitoes was lower than that of CYP6P9a-RS (P<0.004) and of CYP6P9a-RR (P<0.006). This suggests that CYP6P9a gene is inked with the feeding success and blood meal size of An. funestus. However, no correlation was found in the expression of CYP6P9a and that of genes encoding for salivary proteins involved in blood meal process. This study suggests that P450-based metabolic resistance may influence the blood feeding process of Anopheles funestus mosquito and consequently its ability to transmit malaria parasites.


Assuntos
Anopheles/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Insetos/metabolismo , Mosquitos Vetores/metabolismo , Animais , Anopheles/efeitos dos fármacos , Anopheles/genética , Anopheles/parasitologia , Sangue/metabolismo , Camarões , Sistema Enzimático do Citocromo P-450/genética , Comportamento Alimentar , Feminino , Glutationa Transferase/genética , Humanos , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Malária/parasitologia , Malária/prevenção & controle , Malária/transmissão , Mosquitos Vetores/efeitos dos fármacos , Mosquitos Vetores/genética , Mosquitos Vetores/parasitologia , Plasmodium/patogenicidade , Piretrinas/farmacologia , Proteínas e Peptídeos Salivares/metabolismo
9.
Chem Biol Interact ; 324: 109084, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32289290

RESUMO

INTRODUCTION: An imbalance between oxidants and antioxidants in favour of oxidants, potentially leading to damage, is termed oxidative stress. Antioxidants (AO), either enzymatic or non-enzymatic, are the ones that can reduce diverse effects of pro-oxidants such as DNA, proteins and lipids damage. Chalcones (1,3-diaryl-2-propen-1-ones) are open chain flavonoids that are widely biosynthesized in plants. Aim of this study was to test antioxidative potency of 15 chalcones (Chs) in in vitro model in serum (native conditions), so as with exogenously induced oxidative stress. MATERIAL AND METHODS: Oxidative stress was induced in serum samples of healthy individuals with 0.25 mmol/L terc-buthyl-hydroperoxide (TBH), and then we monitored the effects of various concentrations of chalcones on oxidative stress parameters: total antioxidative status (TAS), total oxidative status (TOS), total concentration of sulfhydryl group (SHG) and prooxidative-antioxidative balance (PAB), and calculated prooxidative score, antioxidative score, and oxy score (OS). Nonparametric repeated measures ANOVA (Friedman's test) was used for comparison of antioxidative potency of samples with 15 different chalcones, in a native state and upon TBH influence. Spearman's nonparametric correlation analysis was used for estimation of relation between different parameters. RESULTS: Negative Oxy Score (OS) values for Chs11-15 showed significantly stronger antioxidative potency compared to other investigated chalcones (p < 0.05). Ch11, Ch13 and Ch14 remained with negative OS even after TBH addition, whereas OS of Ch12 and Ch15 became positive, with small nominal values. Samples with Ch11 and Ch13 showed significant negative correlation between TAS and TOS (p < 0.05 for both), but in Ch14 sample the negative correlation existed between TAS and PAB (p < 0.05). CONCLUSION: Lower value of OS (i.e. better antioxidative potency) was noticed in samples with Ch11-Ch15. Electron-donor effects of substituent groups as a structural part of these chalcones could explain its antioxidative capability. Phenolic and methyl groups are responsible for antioxidative ability enhancement of five chalcones with the best activity.


Assuntos
Antioxidantes/farmacologia , Sangue/metabolismo , Chalconas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/síntese química , Antioxidantes/química , Sangue/efeitos dos fármacos , Chalconas/síntese química , Chalconas/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , terc-Butil Hidroperóxido/farmacologia
10.
Opt Express ; 28(4): 5119-5133, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32121739

RESUMO

Multi-spectral imaging enables non-invasive sensing of chemical concentrations in biological tissue based on measurement of optical absorption, but invariably in the presence of high levels of scatter. Absorption is normally inferred from measurement of contrast of biological features, such as the vasculature, and so accuracy is degraded by the poorly characterized modulation-transfer function of the imaging optics and overlying tissue. We report how experimental characterization of the spectral variation of the tissue point-spread function and associated objective speckle pattern can be used to characterize the absorption spectrum and chromophore concentration, with a particular emphasis on determination of the ratio of oxygenated to deoxygenated hemoglobin within blood. Absorption measurements are determined purely by the geometry of the experiment, without degradation due to optical aberrations and associated light scatter. The technique offers enhanced and low-cost determination of in vitro or in vivo chromophore characterizations, including blood-gas analysis.


Assuntos
Absorção de Radiação , Sangue/metabolismo , Lasers , Oximetria/métodos , Espalhamento de Radiação , Animais , Simulação por Computador , Cavalos , Oxigênio/metabolismo , Reprodutibilidade dos Testes
11.
Biochem Biophys Res Commun ; 525(3): 740-746, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32145913

RESUMO

The blood-spinal cord barrier (BSCB) is an effective, tightly-connected tissue that reduces secondary spinal cord injury (SCI) by decreasing blood cell infiltration, inflammation, and neuronal cell death during primary SCI. However, the methods and molecular mechanisms of BSCB openness remain elusive. In the present study, we found that microRNA429 (miR-429) plays a vital role in the opening of the blood-spinal cord. Inhibiting the expression of miR-429 (antagomiR-429) resulted in increased expression levels of the tight junction (TJ) proteins, ZO-1, occludin, and claudin-5, in the BSCB and reduced BSCB permeability. Moreover, overexpression of miR-429 (agomiR-429) had the opposite effect. Krüppel-like factor 6 (KLF6) is a transcription factor of the zinc-finger family. Using RT-qPCR and western blotting, we found that miR-429 can negatively regulate the expression of the KLF6. Co-transfection of KLF6 and miR-429 demonstrated that miR-429 negatively regulates KLF6 to mediate TJ protein expression and BSCB permeability. Based on these results, we suggest that KLF6 may be a downstream target of miR-429, mediating TJ protein expression to regulate the BSCB.


Assuntos
Sangue/metabolismo , Fator 6 Semelhante a Kruppel/metabolismo , MicroRNAs/metabolismo , Medula Espinal/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Humanos , Modelos Biológicos , Permeabilidade , Proteínas de Junções Íntimas/metabolismo
12.
Nat Commun ; 11(1): 1148, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32123170

RESUMO

Late-onset Alzheimer's disease (AD) can, in part, be considered a metabolic disease. Besides age, female sex and APOE ε4 genotype represent strong risk factors for AD that also give rise to large metabolic differences. We systematically investigated group-specific metabolic alterations by conducting stratified association analyses of 139 serum metabolites in 1,517 individuals from the AD Neuroimaging Initiative with AD biomarkers. We observed substantial sex differences in effects of 15 metabolites with partially overlapping differences for APOE ε4 status groups. Several group-specific metabolic alterations were not observed in unstratified analyses using sex and APOE ε4 as covariates. Combined stratification revealed further subgroup-specific metabolic effects limited to APOE ε4+ females. The observed metabolic alterations suggest that females experience greater impairment of mitochondrial energy production than males. Dissecting metabolic heterogeneity in AD pathogenesis can therefore enable grading the biomedical relevance for specific pathways within specific subgroups, guiding the way to personalized medicine.


Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/genética , Apolipoproteínas E/genética , Sangue/metabolismo , Metaboloma/genética , Idoso , Doença de Alzheimer/diagnóstico por imagem , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Estudos de Coortes , Feminino , Genótipo , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Tomografia por Emissão de Pósitrons , Fatores Sexuais
13.
BMC Res Notes ; 13(1): 74, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051015

RESUMO

OBJECTIVES: The number of DNA methylome and RNA transcriptome studies is growing, but investigators have to consider the cell type composition of tissues used. In blood samples, the data reflect the picture of a mixture of different cells. Specialized algorithms can address the cell-type heterogeneity issue. We tested if these corrections are correlated between two heterogeneous datasets. RESULTS: We used methylome and transcriptome datasets derived from a cohort of ten individuals whose blood was sampled at two different timepoints. We examined how the cell composition derived from these omics correlated with each other using "CIBERSORT" for the transcriptome and "estimateCellCounts function" in R for the methylome. The correlation coefficients between the two omic datasets ranged from 0.45 to 0.81 but correlations were minimal between two different timepoints. Our results suggest that a posteriori correction of a mixture of cells present in blood samples is reliable. Using an omic dataset to correct a second dataset for relative fractions of cells appears to be applicable, but only when the samples are simultaneously collected. This could be beneficial when there are difficulties to control the cell types in the second dataset, even when the sample size is limited.


Assuntos
Sangue/metabolismo , Epigenoma , Genômica/métodos , Transcriptoma , Estudos de Coortes , Genômica/normas , Humanos , Reprodutibilidade dos Testes
14.
Nat Commun ; 11(1): 39, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911595

RESUMO

Metabolomics examines the small molecules involved in cellular metabolism. Approximately 50% of total phenotypic differences in metabolite levels is due to genetic variance, but heritability estimates differ across metabolite classes. We perform a review of all genome-wide association and (exome-) sequencing studies published between November 2008 and October 2018, and identify >800 class-specific metabolite loci associated with metabolite levels. In a twin-family cohort (N = 5117), these metabolite loci are leveraged to simultaneously estimate total heritability (h2total), and the proportion of heritability captured by known metabolite loci (h2Metabolite-hits) for 309 lipids and 52 organic acids. Our study reveals significant differences in h2Metabolite-hits among different classes of lipids and organic acids. Furthermore, phosphatidylcholines with a high degree of unsaturation have higher h2Metabolite-hits estimates than phosphatidylcholines with low degrees of unsaturation. This study highlights the importance of common genetic variants for metabolite levels, and elucidates the genetic architecture of metabolite classes.


Assuntos
Sangue/metabolismo , Estudo de Associação Genômica Ampla , Análise Química do Sangue , Estudos de Coortes , Humanos , Metabolômica , Locos de Características Quantitativas , Gêmeos/genética
15.
Nat Biotechnol ; 38(3): 303-308, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31959954

RESUMO

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.


Assuntos
Sangue/metabolismo , Proteoma/química , Proteoma/metabolismo , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Azepinas/administração & dosagem , Azepinas/farmacologia , Células Hep G2 , Humanos , Rim/química , Rim/metabolismo , Fígado/química , Fígado/metabolismo , Pulmão/química , Pulmão/metabolismo , Masculino , Espectrometria de Massas , Camundongos , Especificidade de Órgãos , Panobinostat/administração & dosagem , Panobinostat/farmacologia , Estabilidade Proteica , Ratos , Bibliotecas de Moléculas Pequenas/farmacologia , Baço/química , Baço/metabolismo , Testículo/química , Testículo/metabolismo , Termodinâmica , Triazóis/administração & dosagem , Triazóis/farmacologia , Vemurafenib/administração & dosagem , Vemurafenib/farmacologia
16.
IUBMB Life ; 72(1): 39-44, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31778014

RESUMO

GATA factors play central roles in the programming of blood and cardiac cells during embryonic development. Using the experimentally accessible Xenopus and zebrafish models, we report observations regarding the roles of GATA-2 in the development of blood stem cells and GATA-4, -5, and -6 in cardiac development. We show that blood stem cells develop from the dorsal lateral plate mesoderm and GATA-2 is required at multiple stages. Firstly, GATA-2 is required to make the cells responsive to VEGF-A signalling by driving the synthesis of its receptor, FLK-1/KDR. This leads to differentiation into the endothelial cells that form the dorsal aorta. GATA-2 is again required for the endothelial-to-haematopoietic transition that takes place later in the floor of the dorsal aorta. GATA-2 expression is dependent on BMP signalling for each of these inputs into blood stem cell programming. GATA-4, -5, and -6 work together to ensure the specification of cardiac cells during development. We have demonstrated redundancy within the family and also some evolution of the functions of the different family members. Interestingly, one of the features that varies in evolution is the timing of expression relative to other key regulators such as Nkx2.5 and BMP. We show that the GATA factors, Nkx2.5 and BMP regulate each other and it would appear that what is critical is the mutually supportive network of expression rather than the order of expression of each of the component genes. In Xenopus and zebrafish, the cardiac mesoderm is adjacent to an anterior population of cells giving rise to blood and endothelium. This population is not present in mammals and we have shown that, like the cardiac population, the blood and endothelial precursors require GATA-4, -5, and -6 for their development. Later, blood-specific or cardiac-specific regulators determine the ultimate fate of the cells, and we show that these regulators act cross-antagonistically. Fibroblast growth factor (FGF) signalling drives the cardiac fate, and we propose that the anterior extension of the FGF signalling field during evolution led to the recruitment of the blood and endothelial precursors into the heart field ultimately resulting in a larger four chambered heart. Zebrafish are able to successfully regenerate their hearts after injury. To understand the pathways involved, with a view to determining why humans cannot do this, we profiled gene expression in the cardiomyocytes before and after injury, and compared those proximal to the injury with those more distal. We were able to identify an enhancement of the expression of regulators of the canonical Wnt pathway proximal to the injury, suggesting that changes in Wnt signalling are responsible for the repair response to injury.


Assuntos
Sangue/metabolismo , Diferenciação Celular , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/citologia , Animais , Fatores de Transcrição GATA/genética , Humanos , Miócitos Cardíacos/metabolismo
17.
J Gastroenterol Hepatol ; 35(7): 1196-1200, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31758717

RESUMO

BACKGROUND AND AIM: Detecting blood content in the gastrointestinal tract is one of the crucial applications of capsule endoscopy (CE). The suspected blood indicator (SBI) is a conventional tool used to automatically tag images depicting possible bleeding in the reading system. We aim to develop a deep learning-based system to detect blood content in images and compare its performance with that of the SBI. METHODS: We trained a deep convolutional neural network (CNN) system, using 27 847 CE images (6503 images depicting blood content from 29 patients and 21 344 images of normal mucosa from 12 patients). We assessed its performance by calculating the area under the receiver operating characteristic curve (ROC-AUC) and its sensitivity, specificity, and accuracy, using an independent test set of 10 208 small-bowel images (208 images depicting blood content and 10 000 images of normal mucosa). The performance of the CNN was compared with that of the SBI, in individual image analysis, using the same test set. RESULTS: The AUC for the detection of blood content was 0.9998. The sensitivity, specificity, and accuracy of the CNN were 96.63%, 99.96%, and 99.89%, respectively, at a cut-off value of 0.5 for the probability score, which were significantly higher than those of the SBI (76.92%, 99.82%, and 99.35%, respectively). The trained CNN required 250 s to evaluate 10 208 test images. CONCLUSIONS: We developed and tested the CNN-based detection system for blood content in CE images. This system has the potential to outperform the SBI system, and the patient-level analyses on larger studies are required.


Assuntos
Sangue/diagnóstico por imagem , Sangue/metabolismo , Endoscopia por Cápsula/métodos , Aprendizado Profundo , Intestino Delgado/diagnóstico por imagem , Intestino Delgado/patologia , Redes Neurais de Computação , Área Sob a Curva , Humanos , Intestino Delgado/metabolismo , Curva ROC , Estudos Retrospectivos , Sensibilidade e Especificidade
18.
Asian J Psychiatr ; 47: 101805, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31704595

RESUMO

We summarized the observational studies on the correlation between zinc and attention deficit hyperactivity disorder (ADHD) since 1986, extracted relevant data for meta-analysis to determine the relationship between zinc and ADHD. We searched PubMed, Scopus, Cochrane Library, EMBASE (included EMBASE and Medline), Web of Science and Clinical Trials.gov databases from inception to April 8, 2019. We assessed the blood zinc, hair zinc and ADHD by combined the standardized mean difference (SMD) and 95% confidence interval (CI). Statistical analysis was performed using Stata 14.0. We included 11 studies for meta-analysis. Of these, 8 studies comprising 1311 participants reported blood zinc and 3 studies comprising 206 participants reported hair zinc. The zinc levels in blood (SMD: -0.91, 95% CI: -1.88-0.07, P(SMD) < 0.068), and hair (SMD: 1.42, 95% CI: -4.49-7.33, P(SMD) = 0.638) not significantly compare ADHD with controls. Nevertheless, high heterogeneity (I2 > 97.3%) emerged among the included studies. The subgroup analysis showed that the heterogeneity of samples >100 group was significantly reduced. The sensitivity analysis found that the results changed significantly after excluding the only cross-sectional study. In conclusion, our meta-analysis showed that there was no statistically significant difference in blood zinc and hair zinc levels between ADHD children and adolescents compared with healthy children and adolescents.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Sangue/metabolismo , Cabelo/metabolismo , Zinco/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/sangue , Criança , Pré-Escolar , Feminino , Humanos , Masculino
19.
Drug Alcohol Depend ; 206: 107703, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31785998

RESUMO

BACKGROUND: Little is known about the functional mechanisms through which genetic loci associated with substance use traits ascertain their effect. This study aims to identify and functionally annotate loci associated with substance use traits based on their role in genetic regulation of gene expression. METHODS: We evaluated expression Quantitative Trait Loci (eQTLs) from 13 brain regions and whole blood of the Genotype-Tissue Expression (GTEx) database, and from whole blood of the Depression Genes and Networks (DGN) database. The role of single eQTLs was examined for six substance use traits: alcohol consumption (N = 537,349), cigarettes per day (CPD; N = 263,954), former vs. current smoker (N = 312,821), age of smoking initiation (N = 262,990), ever smoker (N = 632,802), and cocaine dependence (N = 4,769). Subsequently, we conducted a gene level analysis of gene expression on these substance use traits using S-PrediXcan. RESULTS: Using an FDR-adjusted p-value <0.05 we found 2,976 novel candidate genetic loci for substance use traits, and identified genes and tissues through which these loci potentially exert their effects. Using S-PrediXcan, we identified significantly associated genes for all substance traits. DISCUSSION: Annotating genes based on transcriptomic regulation improves the identification and functional characterization of candidate loci and genes for substance use traits.


Assuntos
Usuários de Drogas/psicologia , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Locos de Características Quantitativas/genética , Transtornos Relacionados ao Uso de Substâncias/genética , Sangue/metabolismo , Encéfalo/metabolismo , Perfilação da Expressão Gênica , Humanos , Metanálise como Assunto , Fenótipo , Transtornos Relacionados ao Uso de Substâncias/psicologia , Transcriptoma/genética
20.
Phytomedicine ; 74: 152928, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31451286

RESUMO

BACKGROUND: Quality control of traditional Chinese medicine (TCM) has always been a hot issue to TCM. However, due to the complexity of TCM ingredients, the current quality standards of TCM have problems that are difficult to guarantee clinical efficacy. American ginseng, the dried roots of Pawajc quinquefolium L. (Araliaceae), is a valuable herbal medicine due to various pharmacological effects and huge health benefit, which are associated with numerous active ingredients such as ginsenosides. Although a large number of studies have investigated the active ingredients of American ginseng, Q-markers reflecting comprehensive review on its efficacies has yet been unrevealed. PURPOSE: The study aims to discover the Q-markers of Panax quinquefolius (American ginseng), provides a powerful method to clarify the significant ingredents of TCM and help further discovering extensive quality evaluation model,contributing to a significant improvement of TCM quality standard. METHODS: Mice general status, biochemical indexes assay, urine metabolic profile, and serum metabolic profile were utilized for model replication and efficacy evaluation. The in vitro and in vivo constituents of American ginseng using ultra-high performance liquid chromatography coupled with mass spectrometry (UPLC-MS) with Serum Pharmacochemistry of TCM were in-depth investigated. Q-markers that were associated with core markers of therapeutic effects were excavated by a plotting of correlation between marker metabolites and serum constituents (PCMS) approach. RESULTS: Correlation analysis of 41 blood and urine labeled metabolites with 14 serum components showed that 24-methyl-7-cholesten-3ß-ol, zizybeoside II, betulin, ginsenoside Rd, cinnamyl alcohol, pseudoginsenoside F11 is highly correlated with the therapeutic effects of Compound Zaofan Pill (CZP), while pseudoginsenoside F11 and ginsenoside Rd are highly correlated with the therapeutic effects of American ginseng. The six absorbed blood compounds can be considered as potential Q-markers for compound, of which two compounds, such as pseudoginsenoside F11 and ginsenoside Rd, can be considered as potential Q-markers for American ginseng. CONCLUSION: The study has demonstrated that the Chinmedomics is an effective, comprehensive and fire-new method for discovering the Q-markers of TCM, and it may be more reasonable choices to establish quality standards of TCM.


Assuntos
Biomarcadores Farmacológicos/análise , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Panax/química , Animais , Biomarcadores Farmacológicos/sangue , Biomarcadores Farmacológicos/urina , Sangue/efeitos dos fármacos , Sangue/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/análise , Ginsenosídeos/análise , Espectrometria de Massas , Medicina Tradicional Chinesa/normas , Camundongos , Raízes de Plantas/química , Plantas Medicinais/química , Controle de Qualidade , Urinálise
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