The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs.
The aim of this study is to investigate the effect of osteopontin (OPN) on functional activity of late endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for both 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1). Expression of von Willbrand factor (vWF) and kinase insert domain receptor (KDR) were detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 24 h with OPN (to make a series of final concentration: 0.005 µg/ml, 0.01 µg/ml, 0.05 µg/ml, 0.5 µg/ml, 2.5 µg/ml), or vehicle control. The proliferation, migration, and in vitro vasculogenesis activity of late EPCs were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay and an in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with OPN dose-dependently inhibited the proliferative, adhesive, and in vitro vasculogenesis capacity and increased migratory activity of late EPCs.
A variety of mycotoxins from different sources frequently contaminate farm products, presenting a potential toxicological concern for animals and human. Mycotoxin exposure has been the focus of attention for governments around the world. To date, biomarkers are used to monitor mycotoxin exposure and promote new understanding of their role in chronic diseases. The goal of this research was to develop and validate a sensitive UHPLC-MS/MS method using isotopically-labeled internal standards suitable for accurate determination of 18 mycotoxin biomarkers, including fumonisins, ochratoxins, Alternaria and emerging Fusarium mycotoxins (fumonisin B1, B2, and B3, hydrolyzed fumonisin B1 and B2, ochratoxin A, B, and alpha, alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid, beauvericin, enniatin A, A1, B, and B1) in human urine. After enzymatic digestion with β-glucuronidase, human urine samples were cleaned up using HLB solid phase extraction cartridges prior to instrument analysis. The multi-mycotoxin and analyte-specific method was validated in-house, providing satisfactory results. The method provided good linearity in the tested concentration range (from LOQ up to 25–500 ng/mL for different analytes), with R2 from 0.997 to 0.999. The limits of quantitation varied from 0.0002 to 0.5 ng/mL for all analytes in urine. The recoveries for spiked samples were between 74.0% and 133%, with intra-day precision of 0.5%–8.7% and inter-day precision of 2.4%–13.4%. This method was applied to 60 urine samples collected from healthy volunteers in Beijing, and 10 biomarkers were found. At least one biomarker was found in all but one of the samples. The high sensitivity and accuracy of this method make it practical for human biomonitoring and mycotoxin exposure assessment.
As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood‐borne infections since donated blood is the only source of RBCs. Although large‐scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild‐type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of β‐thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood‐derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell‐cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.
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