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Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs,the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach,as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition,incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results,we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol,which will improve both accuracy and comparability of NGS results across different sample batches,laboratories and NGS platforms. View Publication

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine,by virtue of its polyphenolic constituents,may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene),a polyphenol present in most red wines,on functional and biochemical responses of PMN,upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM,mean+/-s.e.mean),as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM,IC50 18.4+/-1.8 and 31+/-1.8 microM),and C5a (0.1 microM,IC50 41.6+/-3.5 and 42+/-8.3 microM),and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4),6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation,as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb,recognizing an activation-dependent epitope on MAC-1. Consistently,PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results,indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions,may provide some biological plausibility to the protective effect of red wine consumption against CHD. View Publication

Mitochondrial diseases are genetically heterogeneous and present a broad clinical spectrum among patients; in most cases,genetic determinants of mitochondrial diseases are heteroplasmic mitochondrial DNA (mtDNA) mutations. However,it is uncertain whether and how heteroplasmic mtDNA mutations affect particular cellular fate-determination processes,which are closely associated with the cell-type-specific pathophysiology of mitochondrial diseases. In this study,we established two isogenic induced pluripotent stem cell (iPSC) lines each carrying different proportions of a heteroplasmic m.3243A>G mutation from the same patient; one exhibited apparently normal and the other showed most likely impaired mitochondrial respiratory function. Low proportions of m.3243A>G exhibited no apparent molecular pathogenic influence on directed differentiation into neurons and cardiomyocytes,whereas high proportions of m.3243A>G showed both induced neuronal cell death and inhibited cardiac lineage commitment. Such neuronal and cardiac maturation defects were also confirmed using another patient-derived iPSC line carrying quite high proportion of m.3243A>G. In conclusion,mitochondrial respiratory dysfunction strongly inhibits maturation and survival of iPSC-derived neurons and cardiomyocytes; our presenting data also suggest that appropriate mitochondrial maturation actually contributes to cellular fate-determination processes during development. View Publication

Tissue-resident macrophages,such as microglia,Kupffer cells,and Langerhans cells,derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident macrophages self-renew locally,independently of circulating monocytes and HSCs. In contrast,adult blood monocytes,as well as infiltrating,gut,and dermal macrophages,derive from Myb-dependent HSCs. These findings are derived from the mouse,using gene knockouts and lineage tracing,but their applicability to human development has not been formally demonstrated. Here,we use human induced pluripotent stem cells (iPSCs) as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy,we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent,RUNX1-,and SPI1 (PU.1)-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages,such as alveolar and kidney macrophages,microglia,Kupffer cells,and Langerhans cells. View Publication

BACKGROUND Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION Western blot,PCR,immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging,using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated,cell membrane-localized CFTR was detected in non-CF monocytes,being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and,to a lesser extent,obligate CFTR heterozygous carriers (HTZ: 15 subjects),but it failed in monocytes from CF patients (44 subjects). We propose an index,which values in CF patients are significantly (ptextless0.001) lower than in the other two groups. Nasal Potential Difference,measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93,95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials. View Publication