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1.
Nat Protoc ; 16(4): 1966-1994, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33674789

RESUMO

A standardized data workflow is described for large-scale serum metabolomic studies using multisegment injection-capillary electrophoresis-mass spectrometry. Multiplexed separations increase throughput (<4 min/sample) for quantitative determination of 66 polar/ionic metabolites in serum filtrates consistently detected (coefficient of variance (CV) <30%) with high frequency (>75%) from a multi-ethnic cohort of pregnant women (n = 1,004). We outline a validated protocol implemented in four batches over a 7-month period that includes details on preventive maintenance, sample workup, data preprocessing and metabolite authentication. We achieve stringent quality control (QC) and robust batch correction of long-term signal drift with good mutual agreement for a wide range of metabolites, including serum glucose as compared to a clinical chemistry analyzer (mean bias = 11%, n = 668). Control charts for a recovery standard (mean CV = 12%, n = 2,412) and serum metabolites in QC samples (median CV = 13%, n = 202) demonstrate acceptable intermediate precision with a median intraclass coefficient of 0.87. We also report reference intervals for 53 serum metabolites from a diverse population of women in their second trimester of pregnancy.


Assuntos
Eletroforese Capilar/métodos , Eletroforese Capilar/normas , Estudos Epidemiológicos , Ensaios de Triagem em Larga Escala/métodos , Metaboloma , Soro/metabolismo , Glicemia/metabolismo , Calibragem , Canadá , Estudos de Coortes , Jejum/sangue , Feminino , Humanos , Íons , Espectrometria de Massas , Metabolômica , Gravidez , Análise de Componente Principal , Controle de Qualidade , Padrões de Referência , Valores de Referência
2.
Int J Nanomedicine ; 16: 201-211, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33447035

RESUMO

Objective: PEGylated superparamagnetic iron oxide (SPIO) is the most promising alternatives to gadolinium-based contrast agents (GBCAs) in MRI. This paper is to explore the imaging effects of PEGylated SPIO, which is influenced by particle sizes and surface polyethylene glycol (PEG) coating, using as MRI contrast agents at different magnetic field intensities. Methods: Firstly, nine PEGylated monocrystalline SPIO nanoparticles with different nanocrystal sizes and different molecular weights PEG coating were prepared, and then physical and biological properties were analyzed. Finally, MRI imaging in vivo was performed to observe the imaging performance. Results: Nine PEGylated monocrystalline SPIO nanoparticles have good relaxivities, serum stability, and biosecurity. At the same time, they show different imaging characteristics at different magnetic field intensities. Eight-nanometer SPIO@PEG5k is an effective T 2 contrast agent at 3.0 T (r 2/r 1 = 14.0), is an ideal T 1-T 2 dual-mode contrast agent at 1.5 T (r 2/r 1 = 6.52), and is also an effective T 1 contrast agent at 0.5 T (r 2/r 1 = 2.49), while 4-nm SPIO@PEG5k is a T 1-T 2 dual-mode contrast agent at 3.0 T (r 2/r 1 = 5.24), and is a useful T 1 contrast agent at 0.5 T (r 2/r 1 = 1.74) and 1.5 T (r 2/r 1 = 2.85). MRI studies in vivo at 3.0 T further confirm that 4-nm SPIO@PEG5k displays excellent T 1-T 2 dual-mode contrast enhancement, whereas 8-nm SPIO@PEG5k only displays T 2 contrast enhancement. Conclusion: PEGylated SPIOs with different nanocrystal sizes and PEG coating can be used as T 1, T 2, or T 1-T 2 dual-mode contrast agents to meet the clinical demands of MRI at specific magnetic fields.


Assuntos
Meios de Contraste/química , Imagem por Ressonância Magnética , Nanocompostos/química , Polietilenoglicóis/química , Animais , Campos Magnéticos , Masculino , Camundongos , Nanocompostos/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Células RAW 264.7 , Ratos Sprague-Dawley , Soro/metabolismo
3.
Int J Nanomedicine ; 16: 283-296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33469287

RESUMO

Methods: In this study, we used MTT assays to demonstrate that a combination of SPIO-Serum and wild-type p53 overexpression can reduce ovarian cancer cell viability in vitro. Prussian blue staining and iron assays were used to determine changes in intracellular iron concentration following SPIO-Serum treatment. TEM was used to evaluate any mitochondrial damage induced by SPIO-Serum treatment, and Western blot was used to evaluate the expression of the iron transporter and lipid peroxidation regulator proteins. JC-1 was used to measure mitochondrial membrane potential, and ROS levels were estimated by flow cytometry. Finally, xCT protein expression and mitochondrial ROS levels were confirmed using fluorescence microscopy. Results: SPIO-Serum effectively induced lipid peroxidation and generated abundant toxic ROS. It also facilitated the downregulation of GPX4 and xCT, ultimately resulting in iron-dependent oxidative death. These effects could be reversed by iron chelator DFO and lipid peroxidation inhibitor Fer-1. SPIO-Serum treatment disrupted intracellular iron homeostasis by regulating iron uptake and the cells presented with missing mitochondrial cristae and ruptured outer mitochondrial membranes. Moreover, we were able to show that p53 contributed to SPIO-Serum-induced ferroptosis in ovarian cancer cells. Conclusion: SPIO-Serum induced ferroptosis and overexpressed p53 contributed to ferroptosis in ovarian cancer cells. Our data provide a theoretical basis for ferroptosis as a novel cell death phenotype induced by nanomaterials.


Assuntos
Ferroptose , Neoplasias Ovarianas/patologia , Soro/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Neoplasias Ovarianas/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo
4.
PLoS One ; 15(6): e0234145, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497126

RESUMO

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Müller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Müller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Müller cells, under H2O2 and IL-1ß damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1ß, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNF- α), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1ß. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.


Assuntos
Células Ependimogliais/citologia , Células Ependimogliais/efeitos dos fármacos , Sangue Fetal/metabolismo , Soro/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Ependimogliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Polissacarídeos/metabolismo , Ratos , Fator de Necrose Tumoral alfa/metabolismo
5.
Nat Commun ; 11(1): 3186, 2020 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-32581242

RESUMO

Mass spectrometry based metabolomics is a widely used approach in biomedical research. However, current methods coupling mass spectrometry with chromatography are time-consuming and not suitable for high-throughput analysis of thousands of samples. An alternative approach is flow-injection mass spectrometry (FI-MS) in which samples are directly injected to the ionization source. Here, we show that the sensitivity of Orbitrap FI-MS metabolomics methods is limited by ion competition effect. We describe an approach for overcoming this effect by analyzing the distribution of ion m/z values and computationally determining a series of optimal scan ranges. This enables reproducible detection of ~9,000 and ~10,000 m/z features in metabolomics and lipidomics analysis of serum samples, respectively, with a sample scan time of ~15 s and duty time of ~30 s; a ~50% increase versus current spectral-stitching FI-MS. This approach facilitates high-throughput metabolomics for a variety of applications, including biomarker discovery and functional genomics screens.


Assuntos
Análise de Injeção de Fluxo/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Linhagem Celular Tumoral , Análise de Injeção de Fluxo/normas , Ensaios de Triagem em Larga Escala , Humanos , Íons/química , Lipidômica/métodos , Espectrometria de Massas/normas , Metabolômica/normas , Soro/química , Soro/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(19): 10476-10483, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32354992

RESUMO

Cholesterol-laden macrophage foam cells are a hallmark of atherosclerosis. For that reason, cholesterol metabolism in macrophages has attracted considerable scrutiny, particularly the mechanisms by which macrophages unload surplus cholesterol (a process referred to as "cholesterol efflux"). Many studies of cholesterol efflux in macrophages have focused on the role of ABC transporters in moving cholesterol onto high-density lipoproteins (HDLs), but other mechanisms for cholesterol efflux likely exist. We hypothesized that macrophages have the capacity to unload cholesterol directly onto adjacent cells. To test this hypothesis, we used methyl-ß-cyclodextrin (MßCD) to load mouse peritoneal macrophages with [13C]cholesterol. We then plated the macrophages (in the absence of serum or HDL) onto smooth muscle cells (SMCs) that had been metabolically labeled with [15N]choline. After incubating the cells overnight in the absence of HDL or serum, we visualized 13C and 15N distribution by nanoscale secondary ion mass spectrometry (NanoSIMS). We observed substantial 13C enrichment in SMCs that were adjacent to [13C]cholesterol-loaded macrophages-including in cytosolic lipid droplets of SMCs. In follow-up studies, we depleted "accessible cholesterol" from the plasma membrane of [13C]cholesterol-loaded macrophages with MßCD before plating the macrophages onto the SMCs. After an overnight incubation, we again observed substantial 13C enrichment in the SMCs adjacent to macrophages. Thus, macrophages transfer cholesterol to adjacent cells in the absence of serum or HDL. We suspect that macrophages within tissues transfer cholesterol to adjacent cells, thereby contributing to the ability to unload surplus cholesterol.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , Transporte Biológico , Células Espumosas/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Macrófagos/fisiologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Soro/metabolismo , beta-Ciclodextrinas/metabolismo
7.
PLoS One ; 15(4): e0231473, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315325

RESUMO

The aim of this study was to determine the effect of autologous serum (AS) eye drops on the density of human leucocyte antigen (HLA)-DR-positive epithelial cells and Langerhans cells on the ocular surface of patients with bilateral severe dry eye disease (DED) due to graft-versus-host disease (GvHD) or Sjögren's syndrome (SS). The study was conducted on 24 patients (48 eyes). AS was applied 6-10 times daily for 3 months together with regular artificial tear therapy. HLA-DR-positive cells were detected by direct immunocytochemistry on upper bulbar conjunctiva imprints obtained before and after treatment. The application of AS drops led to a statistically significant increase in the mean density of aberrant HLA-DR-positive conjunctival epithelial cells (p < 0.05) and HLA-DR-positive Langerhans cells (p < 0.05) in the GvHD group. Aberrant HLA-DR-positive epithelial cells in the SS group were decreased non-significantly. All patients reported a significant decrease in the Ocular Surface Disease Index (p < 0.01), which indicates improvement of the patient's subjective feelings after therapy. There was an expected but non-significant decrease of aberrant HLA-DR-positive conjunctival epithelial cells in the SS group only. However, the increased density of HLA-DR-positive cells, indicating slight subclinical inflammation, does not outweigh the positive effect of AS in patients with DED from GvHD.


Assuntos
Túnica Conjuntiva/metabolismo , Epitélio/metabolismo , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/metabolismo , Antígenos HLA-DR/metabolismo , Soro/metabolismo , Síndrome de Sjogren/metabolismo , Adulto , Idoso , Contagem de Células/métodos , Túnica Conjuntiva/efeitos dos fármacos , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Soluções Oftálmicas/uso terapêutico , Síndrome de Sjogren/tratamento farmacológico
8.
Zhongguo Zhong Yao Za Zhi ; 45(2): 383-390, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32237322

RESUMO

Enzyme-linked immunosorbent assay(ELISA) and metabolomics were used to analyze and compare two animal models of heart-kidney insomnia, in order to explore a more ideal animal model and preliminarily explore the essence of heart-kidney insomnia. Based on the clinical symptoms and disease characteristics of heart-kidney insomnia, the animal model of heart-kidney insomnia was reproduced through intraperitoneal injection with p-chlorophenylalanine(PCPA) and multi-factor interaction. The animal model of disease-syndrome combination was evaluated by behavioral observation, ELISA and metabolomics. Wistar rats were randomly divided into normal group, PCPA group and compound model group(FH). The rats' behavior, body weight, adrenal index and spleen index were recorded. The levels of corticotropin releasing hormone(CRH) and adrenocorticotropin(ACTH) in serum were detected by ELISA, and the differential metabolites in serum were detected by UPLC-QE-MS. The body weight and adrenal index in FH group were significantly lower than those in PCPA group(P<0.05); whereas ACTH and CRH in FH group were significantly higher than those in PCPA group by ELISA; nine potential biomarkers were identified by serum sample statistics. There were four main metabolic pathways in cardiorenal insomnia: pentose phosphate metabolism, alanine, aspartic acid and glutamic acid metabolism, histidine metabolism, and taurine and subtaurine metabolism. PCPA and multi-factor interaction method can successfully replicate the insomnia model, but multi-factor modeling method is more similar to clinical traditional Chinese medicine syndrome. Animal behavior, ELISA and metabolomics were used to evaluate the rat model of cardiorenal insomnia from in vitro to in vivo, from macro to micro, and from individual to the whole.


Assuntos
Modelos Animais de Doenças , Metaboloma , Soro/metabolismo , Distúrbios do Início e da Manutenção do Sono/metabolismo , Animais , Medicina Tradicional Chinesa , Ratos , Ratos Wistar
9.
J. oral res. (Impresa) ; 9(2): 86-92, abr. 30, 2020. ilus, tab
Artigo em Inglês | LILACS | ID: biblio-1151843

RESUMO

Introduction: Lichen planus is one of the most common oral mucosal lesions. Transforming growth factor-ß (TGF- ß) has a marked effect on epithelial­mesenchymal transition and immune cells function. Vascular Endothelial Growth Factor (VEGF) is a key regulator of vasculogenesis and angiogenesis. Tumor necrosis factor-α (TNF-α) mediates T-lymphocyte homing and apoptosis of epithelial cells. Objetive: The present study was conducted in order to compare the expression of serum and salivary TGF- ß, VEGF, TNF-α between OLP patients and control individuals to investigate if saliva can be used as an alternative to serum for diagnostic purposes and for monitoring disease. Materials and Methods: 23 OLP patients and 23 control individuals were included to evaluate serum and salivary TGF-ß, VEGF, TNF-α using ELISA kits. Five milliliters of venous blood was collected and unstimulated saliva was collected by the spitting method. Results: Serum and salivary levels of TGF- ß, VEGF, TNF-α are higher in OLP patients compared to normal controls. Mean difference is higher in saliva than serum. Moreover, there was a significant difference in serum and salivary VEGF and TNF-α between symptomatic and asymptomatic groups. Conclusions: Saliva can be a used as a substitute for serum to evaluate levels of the assessed biomarkers.


Introducción: El liquen plano oral es una de las lesiones de la mucosa oral más comunes. El factor de crecimiento transformante ß (TGF-ß) tiene un efecto marcado sobre la transición epitelial-mesenquimal y la función de las células inmunes. El factor de crecimiento endotelial vascular (VEGF) es un regulador clave de la vasculogénesis y la angiogénesis. El factor de necrosis tumoral α (TNF-α) media la localización de los linfocitos T y la apoptosis de las células epiteliales. Objetivo: El presente estudio se realizó con el fin de comparar la expresión en suero y saliva de TGF-ß, VEGF, TNF-α entre pacientes con OLP y personas de control para investigar si la saliva se puede utilizar como alternativa al suero para fines de diagnóstico y monitoreo de la enfermedad. Material y Métodos: Se incluyeron 23 pacientes con OLP y 23 individuos control para evaluar los niéveles en suero y en saliva de TGF- ß, VEGF, TNF-α utilizando kits ELISA. Se recogieron cinco mililitros de sangre venosa y se recogió saliva no estimulada por el método de escupir. Resultado: Los niveles séricos y salivales de TGF-ß, VEGF, TNF-α son más altos en pacientes con OLP en comparación con los controles normales. La diferencia media es mayor en saliva que en suero. Además, hubo una diferencia significativa de VEGF y TNF-α en suero y saliva entre los grupos sintomáticos y asintomáticos. Conclusion: La saliva puede usarse como un sustituto del suero para evaluar los niveles de los biomarcadores estudiados


Assuntos
Humanos , Masculino , Feminino , Saliva/metabolismo , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfa , Líquen Plano Bucal/diagnóstico , Soro/metabolismo , Fator A de Crescimento do Endotélio Vascular , Egito , Mucosa Bucal , Necrose
10.
Biochem Biophys Res Commun ; 525(3): 626-632, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32122653

RESUMO

BACKGROUND: When proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. PURPOSE: Since persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. RESULTS: -a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H2O2, in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates (SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 µM H2O2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H2O2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE: - These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H2O2).


Assuntos
Ferroptose/efeitos dos fármacos , Melanoma Experimental/patologia , Nitroprussiato/farmacologia , Soro/metabolismo , Animais , Butadienos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobalto/farmacologia , Meios de Cultura Livres de Soro , Ciclina A/metabolismo , Peróxido de Hidrogênio/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Nitrilos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transferrina/deficiência , Transferrina/metabolismo
11.
Sci Rep ; 10(1): 5651, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221384

RESUMO

The purpose of this study is to evaluate the prognostic value of preoperative Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA+-M2BP) in predicting overall survival for patients with hepatitis B- and hepatitis C-related early-stage hepatocellular carcinoma (ESHCC) after liver resection. Post-operative survival rates were compared according to WFA+-M2BP level and tumor stage. Six hundred and ten patients were identified and 198 were removed after application of the exclusion criteria; the median follow-up time was 4.33 years, and cancer-related death occurred in 117 (28.4%) patients. Age (p = 0.03), fibrosis grade (p = 0.042), cancer stage (p = 0.01), and WFA+-M2BP level (p = 0.001) were identified as independent risk factors for poor overall survival. The overall survival rates at 3 and 5 years for patients with WFA+-M2BP ≤ 1.12 were 0.92 and 0.90, respectively, and 0.76 and 0.61 for patients with WFA+-M2BP > 1.12 (p < 0.001). During the analysis of survival prediction, serum WFA+-M2BP level exhibited a higher log-likelihood and a lower AIC value compared to TNM stage (log likelihood: -638; AIC: 1279). Pre-operative serum WFA+-M2BP level provided important prognostic information after curative hepatic resection in our study.


Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Lectinas de Plantas/metabolismo , Receptores de N-Acetilglucosamina/metabolismo , Soro/metabolismo , Idoso , Feminino , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias/métodos , Prognóstico , Taxa de Sobrevida
12.
Oxid Med Cell Longev ; 2020: 5363546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32064026

RESUMO

The present study was performed to evaluate the antioxidant and intestinal protective effects of baicalin-copper on deoxynivalenol-challenged piglets. Forty weaned piglets were randomly divided into four groups and assigned to different diets: (1) basal diet (Con), (2) 4 mg/kg deoxynivalenol of basal diet (DON), (3) 5 g/kg baicalin-copper of basal diet (BCU); and (4) 4 mg/kg deoxynivalenol + 5 g/kg baicalin-copper of basal diet (DBCU). The results showed that the ADFI and ADG of piglets in the DON group were markedly lower than those in the Con group, but the ADFI and ADG of the DBCU group were not significantly different from those of the Con group. In piglets fed a DON-contaminated diet, dietary supplementation with BCU significantly decreased the mRNA levels of P70S6K, 4E-BP1, and HSP70 in the liver, the protein expression of HO-1 in the jejunum, and the expression of p-Nrf2 and p-NF-κB in the ileum but increased Mn-SOD activity in serum. Dietary supplementation with BCU increased jejunal maltase, ZIP4 and MT mRNA levels, and serum concentrations of Arg, Val, Ile, Leu, Lys, and Tyr in DON-contaminated piglets. In summary, BCU can alleviate the growth impairment induced by DON and enhance antioxidant capacity and nutrition absorption in piglets fed DON-contaminated diets.


Assuntos
Antioxidantes/metabolismo , Flavonoides/farmacologia , Íleo/efeitos dos fármacos , Jejuno/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tricotecenos/toxicidade , Aminoácidos/sangue , Ração Animal , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cobre/química , Dieta , Suplementos Nutricionais , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Íleo/metabolismo , Jejuno/citologia , Jejuno/enzimologia , Jejuno/metabolismo , Fígado/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Estresse Oxidativo/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Soro/enzimologia , Soro/metabolismo , Superóxido Dismutase-1/sangue , Suínos , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismo
13.
Sci Rep ; 10(1): 3056, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080297

RESUMO

Toll-like receptor 4 (TLR4) and one of its endogenous ligands myeloid-related protein 8 (MRP8 or S100A8), especially expressed in macrophages, play an important role in diabetic nephropathy and autoimmune disorders. However, detailed mechanisms and consequence of MRP8 expression remain unknown, partly due to embryonic lethality of MRP8 knockout mice. In this study, Myeloid lineage cell-specific MRP8 knockout mice were generated, and nephrotoxic serum-induced glomerulonephritis was developed. Mice with conditional ablation of MRP8 gene in myeloid cells exhibited less severe histological damage, proteinuria and inflammatory changes compared to control mice. Mechanism of MRP8 upregulation was investigated using cultured cells. Co-culture of macrophages with mesangial cells or mesangial cell-conditioned media, but not with proximal tubules, markedly upregulated MRP8 gene expression and inflammatory M1 phenotype in macrophages, which was attenuated in MRP8-deleted bone marrow-derived macrophages. Effects of MRP8 deletion was further studied in the context of macrophage-inducible C-type lectin (Mincle), which is critically involved in maintenance of M1 phenotype of macrophages. MRP8 ablation in myeloid cells suppressed the induction of Mincle expression on macrophages in glomerulonephritis. Thus, we propose that intraglomerular crosstalk between mesangial cells and macrophages plays a role in inflammatory changes in glomerulonephritis, and MRP8-dependent Mincle expression in macrophage may be involved in the process.


Assuntos
Calgranulina A/metabolismo , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomérulos Renais/patologia , Macrófagos/metabolismo , Células Progenitoras Mieloides/metabolismo , Soro/metabolismo , Animais , Calgranulina A/deficiência , Linhagem da Célula , Modelos Animais de Doenças , Deleção de Genes , Integrases/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/patologia , Proteínas de Membrana/metabolismo , Células Mesangiais/metabolismo , Camundongos , Camundongos Knockout , Células RAW 264.7 , Recombinação Genética/genética , Fibras de Estresse/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima
14.
Cryobiology ; 92: 208-214, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32004575

RESUMO

The freeze-thaw procedure causes irreversible structural and functional changes in human spermatozoa. In order to decrease the detrimental effects of cryopreservation and improve the quality of post-thawed spermatozoa, the constituents of the freezing solution attracted considerable attention. In this study, for the first time, we evaluated the efficacy of knockout serum replacement (KSR) as a substitute for human serum albumin (HSA) for cryopreservation of human spermatozoa. Twenty semen samples were collected from normozoospermic men and divided them into five equal groups. One of the aliquots was diluted with glycerol-based medium as a control group (CON). The other four aliquots were diluted with the sucrose solution containing 5% HSA (H5), 10% HSA (H10), 5% KSR (K5), and 10% KSR (K10). The diluted samples were frozen and preserved in liquid nitrogen. Post thawed sperm parameters including motion characteristics, viability, membrane integrity, mitochondrial activity, acrosome integrity and DNA intactness in all of the sucrose-based groups were comparable with glycerol-based medium. The replacement of HSA by 10% KSR in the freezing medium resulted in significantly higher post-thawed viability, acrosome integrity and DNA intactness compared with other sucrose-based groups. In conclusion, the addition of 10% KSR to the sucrose-based freezing solution improves the quality of post-thawed human spermatozoa and may have potential to develop chemically defined freezing medium.


Assuntos
Criopreservação/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Soro/metabolismo , Acrossomo/efeitos dos fármacos , Adulto , Animais , Congelamento , Glicerol/farmacologia , Humanos , Masculino , Mitocôndrias/efeitos dos fármacos , Motilidade Espermática/efeitos dos fármacos , Sacarose/farmacologia
15.
Int J Mol Sci ; 21(3)2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-32012941

RESUMO

The remarkable success of immune checkpoint inhibitors (ICIs) has given hope of cure for some patients with advanced cancer; however, the fraction of responding patients is 15-35%, depending on tumor type, and the proportion of durable responses is even smaller. Identification of biomarkers with strong predictive potential remains a priority. Until now most of the efforts were focused on biomarkers associated with the assumed mechanism of action of ICIs, such as levels of expression of programmed death-ligand 1 (PD-L1) and mutation load in tumor tissue, as a proxy of immunogenicity; however, their performance is unsatisfactory. Several assays designed to capture the complexity of the disease by measuring the immune response in tumor microenvironment show promise but still need validation in independent studies. The circulating proteome contains an additional layer of information characterizing tumor-host interactions that can be integrated into multivariate tests using modern machine learning techniques. Here we describe several validated serum-based proteomic tests and their utility in the context of ICIs. We discuss test performances, demonstrate their independence from currently used biomarkers, and discuss various aspects of associated biological mechanisms. We propose that serum-based multivariate proteomic tests add a missing piece to the puzzle of predicting benefit from ICIs.


Assuntos
Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Proteômica/métodos , Humanos , Espectrometria de Massas , Análise Multivariada , Neoplasias/metabolismo , Soro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos
16.
BMC Res Notes ; 13(1): 39, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969182

RESUMO

OBJECTIVE: Currently, the replacement of fetal calf serum (FCS) by a more suitable alternative is a sought aim in the field of tissue and cell culture research. Autologous plasma (AP) and especially autologous serum (AS) have been shown to be effective substitutes of FCS in culture media for some of the cell types. Nevertheless, there is no comparative data on the most appropriate supplement for cell media in neutrophil studies, it is now unclear whether AP have a relatively equal, superior or inferior performance to FCS in neutrophil cell culture. In the present study, human blood neutrophils were isolated and cultured in FCS- or AP-supplemented medium. After 12, 36 and 60 h of incubation, cell viability, oxidative burst and CD11b expression were determined by flow cytometry. RESULTS: Compared to the culture of neutrophils in FCS 10% medium, the culture of neutrophils in a medium with AP 10% could prolong their life span without affecting their function. The findings introduce AP as a better supplement for human neutrophil cell culture than FCS and propose a simple and economical procedure for neutrophil isolation and culture.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Neutrófilos/citologia , Plasma/metabolismo , Soro/metabolismo , Apoptose , Antígeno CD11b/metabolismo , Separação Celular , Sobrevivência Celular , Humanos , Explosão Respiratória
17.
Cryobiology ; 92: 255-257, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31954710

RESUMO

To enrich bovine gonocytes from cryopreserved testicular tissues, the cryoprotection effects of the freezing media containing knockout serum replacement (KSR) were examined. Using Minimum essential medium (MEM) + 10% dimethyl sulfoxide (Me2SO) as the basic medium, calf testicular tissues were cryopreserved in media containing 0, 5, 10, 20, 40, 90% KSR and 5% fetal bovine serum (FBS) respectively. Morphologically, the seminiferous cords and interstitium were well preserved in all groups. The gonocytes were all glial cell line-derived neurotrophic factor (GDNF) family receptor α-1 (GFRα-1) positive. The recovery rates in all KSR groups were higher than that of the 10% Me2SO group, while comparable to the 5% FBS group. The enriched gonocytes expressed gonocyte marker GFRα-1 typically. Collectively, supplementation of 5-10% KSR can achieve comparable cryoprotective effects with using 5% FBS, which is useful in future study due to its defined formulation that is more consistent in quality and stable in supply.


Assuntos
Criopreservação/métodos , Crioprotetores/farmacologia , Soro/metabolismo , Testículo/citologia , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/farmacologia , Congelamento , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Masculino , Soro/química
18.
Methods Mol Biol ; 2102: 129-147, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989552

RESUMO

Immunoassay is one of the most commonly used biomedical techniques to detect the expression of an antibody or an antigen in a test sample. Enzyme-linked immunosorbent assay (ELISA) has been used for a variety of applications including diagnostic tools and quality controls. However, one of the main limitations of ELISA is its lack of multiplexing ability, so ELISA may not be an efficient diagnostic tool when a measurement of multiple determinants is needed for samples with limited quantity such as blood or biological samples from newborns or babies. Although similar to ELISA in assay measurement, multiplex platforms such as bead-based Luminex and multi-array-based MSD (Meso Scale Discovery) are widely used to measure multiple biomarkers from a single analysis. Luminex is a xMAP-based technology that combines several different technologies to provide an efficient and accurate measurement of multiple analytes from a single sample. The multiplexing can be achieved because up to 100 distinct Luminex color-coded microsphere bead sets can be coated with a reagent specific to a particular bioassay, allowing the capture and detection of specific analytes from a sample. Using Multi-array and electrochemiluminescence technologies, the MSD platform provides the multiplex capability with similar consistence as observed in ELISA. Various biological samples that can be analyzed by both Luminex and MSD systems include serum, plasma, tissue and cell lysate, saliva, sputum, and bronchoalveolar Lavage (BAL). The most common Luminex and MSD-based assays are to detect a combined set of cytokines to provide a measurement of cytokine expression profiling for a diagnostic purpose.


Assuntos
Anticorpos/imunologia , Biomarcadores/sangue , Imunoensaio/instrumentação , Imunoensaio/métodos , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Fluorescência , Humanos , Pulmão/imunologia , Pulmão/metabolismo , Camundongos , Plasma/imunologia , Plasma/metabolismo , Kit de Reagentes para Diagnóstico , Soro/imunologia , Soro/metabolismo , Fluxo de Trabalho
19.
Analyst ; 145(4): 1103-1109, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-31894758

RESUMO

This mini-review summarizes analytical methods in use to uncover biochemical transformations that magnetic nanoparticles (MNPs) are possibly undergoing while residing in human blood. Examples from the recent literature are presented to illustrate what analytical challenges are to be addressed to shed light on this important issue of biomedical application of MNPs.


Assuntos
Técnicas de Química Analítica/métodos , Imãs/química , Nanopartículas/química , Soro/metabolismo , Humanos
20.
Biol Pharm Bull ; 43(2): 306-318, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31761827

RESUMO

This study focused on the differential metabolomic effects between water extracts of Polygoni Multiflori Radix and Polygoni Multiflori Radix Preparata in rats. The extracts were subsequently administered for 28 d. Serum biochemical indicators were tested, hematoxylin-eosin staining and immunohistochemistry staining were used to detect histopathological changes in the livers. Ultra-performance LC/quadrupole time-of-flight mass spectrometry was used to detect the changes in endogenous metabolites. Finally, we performed detailed analysis of the changes in metabolic pathways. Hematoxylin-eosin staining and immunohistochemistry staining results indicated that the water extracts of Polygoni Multiflori Radix and Polygoni Multiflori Radix Preparata had mild liver injury effect. Fifty-two differential endogenous biomarkers were confirmed as potential biomarkers between Polygoni Multiflori Radix and Polygoni Multiflori Radix Preparata groups. In the positive ion mode, the biomarkers included 31 Phosphatidyl cholines (PCs), six lysoPCs, and ceramide. In the negative ion mode, 12 biomarkers were confirmed, including glycodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid, etc. In Hydrophilic Interaction Liquid Chromatography (HILIC) mode, nine biomarkers were confirmed, including niacinamide, L-palmitoylcarnitine, and butyrylcarnitine, etc. Using MetaboAnalyst 4.0, six related metabolic pathways, including taurine and hypotaurine metabolism, sphingolipid metabolism, glycerophospholipid metabolism, nicotinate and nicotinamide metabolism, arginine and proline metabolism, and tryptophan metabolism and primary bile synthesis, were confirmed as the most differential pathways between the Polygoni Multiflori Radix and Polygoni Multiflori Radix Preparata groups.


Assuntos
Medicamentos de Ervas Chinesas/efeitos adversos , Metabolômica , Polygonum , Soro/metabolismo , Animais , Biomarcadores/análise , Doença Hepática Induzida por Substâncias e Drogas , Cromatografia Líquida de Alta Pressão , Fígado/efeitos dos fármacos , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas , Ratos , Ratos Sprague-Dawley
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