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Sample records for human foetal phenotype

  1. Phenotypic characterization of stem cell factor-dependent human foetal liver-derived mast cells.

    PubMed Central

    Nilsson, G; Forsberg, K; Bodger, M P; Ashman, L K; Zsebo, K M; Ishizaka, T; Irani, A M; Schwartz, L B

    1993-01-01

    Human foetal liver cells are an enriched source of mast cell progenitors that complete their differentiation and mature in response to stem cell factor, the ligand for Kit, in liquid culture. These mast cells are Kit+, metachromatic with toluidine blue+, tryptase+, histamine+ and show ultrastructure features of mast cells. Using a panel of monoclonal antibodies (mAb) against different cell-surface antigens (33 mAb were used), the cell-surface phenotype of human stem cell factor-dependent foetal liver-derived mast cells was examined by flow cytometry. Consistent with previous reports on tissue-derived mast cells, those derived from foetal liver in vitro expressed HLA class I, CD9, CD29, CD33, CD43, CD45 and Kit. Unlike mast cells dispersed from tissue, a high expression of CD13 was found. Also, these in vitro-derived mast cells express little, if any, high-affinity IgE receptor. However, small amounts of mRNA for the alpha-chain in foetal liver-derived mast cells compared to KU812 cells (a human basophil-like cell line) could be detected by Northern blotting. Full expression of Fc epsilon RI may require additional growth factor(s). Images Figure 2 PMID:7688344

  2. Derivation of a novel undifferentiated human foetal phenotype in serum-free cultures with BMP-2

    PubMed Central

    Mirmalek-Sani, Sayed-Hadi; Stokes, Paula J; Tare, Rahul S; Ralph, Esther J; Inglis, Stefanie; Hanley, Neil A; Houghton, Franchesca D; Oreffo, Richard OC

    2009-01-01

    Skeletal stem and progenitor populations provide a platform for cell-based tissue regeneration strategies. Optimized conditions for ex vivo expansion will be critical and use of serum-free culture may allow enhanced modelling of differentiation potential. Maintenance of human foetal femur-derived cells in a chemically defined medium (CDM) with activin A and fibroblast growth factor-2 generated a unique undifferentiated cell population in comparison to basal cultures, with significantly reduced amino acid depletion, appearance and turnover, reduced alkaline phosphatase (ALP) activity and loss of type I and II collagen expression demonstrated by fluorescence immunocytochemistry. Microarray analysis demonstrated up-regulation of CLU, OSR2, POSTN and RABGAP1 and down-regulation of differentiation-associated genes CRYAB, CSRP1, EPAS1, GREM1, MT1X and SRGN as validated by quantitative real-time polymerase chain reaction. Application of osteogenic conditions to CDM cultures demonstrated partial rescue of ALP activity. In contrast, the addition of bone morphogenetic protein-2 (BMP-2) resulted in reduced ALP levels, increased amino acid metabolism and, strikingly, a marked shift to a cobblestone-like cellular morphology, with expression of SOX-2 and SOX-9 but not STRO-1 as shown by immunocytochemistry, and significantly altered expression of metabolic genes (GFPT2, SC4MOL and SQLE), genes involved in morphogenesis (SOX15 and WIF1) and differentiation potential (C1orf19, CHSY-2,DUSP6, HMGCS1 and PPL). These studies demonstrate the use of an intermediary foetal cellular model for differentiation studies in chemically defined conditions and indicate the in vitro reconstruction of the mesenchymal condensation phenotype in the presence of BMP-2, with implications therein for rescue studies, screening assays and skeletal regeneration research. PMID:19438813

  3. Foetal bovine serum-derived exosomes affect yield and phenotype of human cardiac progenitor cell culture

    PubMed Central

    Angelini, Francesco; Ionta, Vittoria; Rossi, Fabrizio; Miraldi, Fabio; Messina, Elisa; Giacomello, Alessandro

    2016-01-01

    Introduction: Cardiac progenitor cells (CPCs) represent a powerful tool in cardiac regenerative medicine. Pre-clinical studies suggest that most of the beneficial effects promoted by the injected cells are due to their paracrine activity exerted on endogenous cells and tissue. Exosomes are candidate mediators of this paracrine effects. According to their potential, many researchers have focused on characterizing exosomes derived from specific cell types, but, up until now, only few studies have analyzed the possible in vitro effects of bovine serum-derived exosomes on cell proliferation or differentiation. Methods: The aim of this study was to analyse, from a qualitative and quantitative point of view, the in vitro effects of bovine serum exosomes on human CPCs cultured either as cardiospheres or as monolayers of cardiosphere-forming cells. Results: Effects on proliferation, yield and molecular patterning were detected. We show, for the first time, that exogenous bovine exosomes support the proliferation and migration of human cardiosphere-forming cells, and that their depletion affects cardiospheres formation, in terms of size, yield and extra-cellular matrix production. Conclusion: These results stress the importance of considering differential biological effects of exogenous cell culture supplements on the final phenotype of primary human cell cultures.

  4. Isolation and in vitro chondrogenic potential of human foetal spine cells.

    PubMed

    Quintin, Aurelie; Schizas, Constantin; Scaletta, Corinne; Jaccoud, Sandra; Gerber, Stefan; Osterheld, Maria-Chiara; Juillerat, Lucienne; Applegate, Lee Ann; Pioletti, Dominique P

    2009-08-01

    Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration. PMID:19413893

  5. [Zonal centrifuge purification of human rabies vaccine obtained on bovine foetal kidney cells (author's transl)].

    PubMed

    Atanasiu, P; Tsiang, H; Lavergne, M; Chermann, J C

    1977-01-01

    A human rabies vaccine is prepared on bovine foetal kidney cells in absence of serum. This vaccine is concentrated and purified by zonal centrifugation. An immunogenic vaccine is obtained from the purified viral particles. Preliminary results are reported. PMID:563208

  6. Adipogenic potential in human mesenchymal stem cells strictly depends on adult or foetal tissue harvest.

    PubMed

    Ragni, Enrico; Viganò, Mariele; Parazzi, Valentina; Montemurro, Tiziana; Montelatici, Elisa; Lavazza, Cristiana; Budelli, Silvia; Vecchini, Alba; Rebulla, Paolo; Giordano, Rosaria; Lazzari, Lorenza

    2013-11-01

    Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Wharton's jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes. PMID:23942228

  7. Diversified Expression of NG2/CSPG4 Isoforms in Glioblastoma and Human Foetal Brain Identifies Pericyte Subsets

    PubMed Central

    Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  8. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture

    PubMed Central

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide

    2016-01-01

    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45−Ter119−Dlk1+ LPCs derived from murine foetal livers formed ALBUMIN (ALB)+CYTOKERATIN (CK)19− non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB−CK19+ cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo. PMID:27335264

  9. Effect of maternal undernutrition on human foetal pancreas morphology in second trimester of pregnancy

    PubMed Central

    Kumar, P. Uday; Ramalaxmi, B.A.; Venkiah, K.; Sesikeran, B.

    2013-01-01

    Background & objectives: Maternal undernutrition and hyperglycaemia during pregnancy, as well as foetal undernutrition affecting the development of foetal endocrine pancreas structure and function, especially that of β-cells is well known. This study was undertaken to look into the changes in pancreatic islets morphology of aborted normal human foetuses (16-20 wk old) of undernourished and adequately nourished mothers. Methods: Foetuses were collected over a 24 month period from medically terminated pregnancies of six undernourished mothers (BMI <18.5 kg/m2) and eight adequately nourished mothers (BMI >18.5 kg/m2). The sections were stained with haematoxylin & eosin as well as Masson trichrome for morphometric estimates such as islet count, area, volume, etc. and immunohistochemistry analysis of β-cells for insulin presence was done. Results: Significant correlations between maternal and foetal parameters were seen. However, there were no statistically significant differences in the number, size or density and beta cell counts of the pancreas among foetal pancreas of mothers with BMI <18.5 and >18.5 kg/m2. Interpretation & conclusions: Our findings indicate that nutritional status of the mother may not have profound influence on the morphology of beta cells of foetal pancreas in second trimester of pregnancy. Further studies need to be done to confirm these findings. PMID:23563373

  10. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed Central

    Speirs, V.; Ray, K. P.; Freshney, R. I.

    1991-01-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts. Images Figure 5 PMID:1654985

  11. Quantitative analysis of miRNA expression in seven human foetal and adult organs.

    PubMed

    Tang, Yanping; Liu, Dong; Zhang, Lijie; Ingvarsson, Sigurdur; Chen, Huiping

    2011-01-01

    miRNAs have been found to repress gene expression at posttranscriptional level in cells. Studies have shown that expression of miRNAs is tissue-specific and developmental-stage-specific. The mechanism behind this could be explained by miRNA pathways. In this study, totally 54 miRNAs were analysed in 7 matched human foetal and adult organs (brain, colon, heart, kidney, liver, lung and spleen) using real-time PCR. Quantitative analysis showed that a big proportion of the 54 miRNAs have higher general expression in the organs of the foetal period than the adult period, with the exception of the heart. The miRNA gene promoter methylation level in the adult stages was higher than in the foetal stages. Moreover, there is a high general expression level of several miRNAs in both stages of brain, kidney, liver, lung and spleen, but not seen in colon and heart. Our results indicate that the miRNAs may play a bigger role in the foetal stage than the adult stage of brain, colon, kidney, liver, lung and spleen. The majority of the miRNAs analysed may play an important role in the growth and development of brain, kidney, liver, lung and spleen. However, a minority of the miRNAs may be functional in colon and heart. PMID:22194897

  12. Transport of the subclasses of human IgG across the yolk-sac of the foetal rabbit

    PubMed Central

    Hemmings, W. A.

    1974-01-01

    The four subclasses of human IgG were labelled either with 125I or 131I and injected into rabbit uteri (24 days pregnant) in pairs. Transmission to the foetal circulation was measured 1 day later. It was found that all four sub-classes are transmitted, though IgG1 enters most readily. Whole labelled human IgG fast and slow electrophoretic fractions prepared by DEAE chromatography were also injected and an isoelectrofocusing analysis carried out on the injected IgG and the foetal serum. There was considerable variation of transmission even between adjacent peaks of the IEF pattern. PMID:4435834

  13. Acetylcholine output and foetal vascular resistance of human perfused placental cotyleda.

    PubMed Central

    Boura, A. L.; Gude, N. M.; King, R. G.; Walters, W. A.

    1986-01-01

    The foetal villous vessels of single cotyleda of human placentae have been perfused with a constant flow of Krebs solution, recording inflow pressure and passing the venous perfusate in cascade over guinea-pig ileum and rat stomach strip preparations in vitro. Each cotyledon released for at least 4 h a substance that was probably acetylcholine. The perfusate caused contractions of both preparations which were inhibited by atropine or hyoscine and potentiated by physostigmine. Contractile activity was destroyed after incubation at 37 degrees C of perfusate with acetylcholinesterase but not with acetylcholinesterase plus physostigmine. When the perfusion temperature was lowered to 34 degrees C or below, acetylcholine output was reduced, the extent depending on the fall in temperature. No change in resistance of the villous vessels occurred during the changes in temperature or in the presence at 37 degrees C of atropine, hyoscine, hexamethonium, (+)-tubocurarine, hemicholinium-3 or bretylium. Submaximal vasoconstrictor responses of the villous vessels to the thromboxane A2-mimetic U46619 were not affected by reduction of the perfusion temperature to 30 degrees C, which lowered acetylcholine-like output by approximately 70%. Responses to U46619, at 37 degrees C, were unchanged during the presence of atropine or hyoscine. Acetylcholine is released into the foetal circulation of the human placenta but no evidence could be obtained that it affects villous vascular smooth muscle tone or vasoconstrictor responses. PMID:3730696

  14. You are what your mother eats: evidence for maternal preconception diet influencing foetal sex in humans

    PubMed Central

    Mathews, Fiona; Johnson, Paul J; Neil, Andrew

    2008-01-01

    Facultative adjustment of sex ratios by mothers occurs in some animals, and has been linked to resource availability. In mammals, the search for consistent patterns is complicated by variations in mating systems, social hierarchies and litter sizes. Humans have low fecundity, high maternal investment and a potentially high differential between the numbers of offspring produced by sons and daughters: these conditions should favour the evolution of facultative sex ratio variation. Yet little is known of natural mechanisms of sex allocation in humans. Here, using data from 740 British women who were unaware of their foetus's gender, we show that foetal sex is associated with maternal diet at conception. Fifty six per cent of women in the highest third of preconceptional energy intake bore boys, compared with 45% in the lowest third. Intakes during pregnancy were not associated with sex, suggesting that the foetus does not manipulate maternal diet. Our results support hypotheses predicting investment in costly male offspring when resources are plentiful. Dietary changes may therefore explain the falling proportion of male births in industrialized countries. The results are relevant to the current debate about the artificial selection of offspring sex in fertility treatment and commercial ‘gender clinics’. PMID:18430648

  15. Biological effects of in vitro THz radiation exposure in human foetal fibroblasts.

    PubMed

    De Amicis, Andrea; Sanctis, Stefania De; Cristofaro, Sara Di; Franchini, Valeria; Lista, Florigio; Regalbuto, Elisa; Giovenale, Emilio; Gallerano, Gian Piero; Nenzi, Paolo; Bei, Roberto; Fantini, Massimo; Benvenuto, Monica; Masuelli, Laura; Coluzzi, Elisa; Cicia, Cristina; Sgura, Antonella

    2015-11-01

    In recent years, terahertz (THz) radiation has been widely used in a variety of applications: medical, security, telecommunications and military areas. However, few data are available on the biological effects of this type of electromagnetic radiation and the reported results, using different genetic or cellular assays, are quite discordant. This multidisciplinary study focuses on potential genotoxic and cytotoxic effects, evaluated by several end-points, associated with THz radiation. For this purpose, in vitro exposure of human foetal fibroblasts to low frequency THz radiation (0.1-0.15THz) was performed using a Compact Free Electron Laser. We did not observe an induction of DNA damage evaluated by Comet assay, phosphorylation of H2AX histone or telomere length modulation. In addiction, no induction of apoptosis or changes in pro-survival signalling proteins were detected. Moreover, our results indicated an increase in the total number of micronuclei and centromere positive micronuclei induction evaluated by CREST analysis, indicating that THz radiation could induce aneugenic rather than clastogenic effects, probably leading to chromosome loss. Furthermore, an increase of actin polymerization observed by ultrastructural analysis after THz irradiation, supports the hypothesis that an abnormal assembly of spindle proteins could lead to the observed chromosomal malsegregation. PMID:26520385

  16. Morphology of the interstitial cells of Cajal of the human ileum from foetal to neonatal life

    PubMed Central

    Faussone-Pellegrini, Maria-Simonetta; Vannucchi, Maria-Giuliana; Alaggio, Rita; Strojna, Aleksandra; Midrio, Paola

    2007-01-01

    Abstract The so-called interstitial cells of Cajal myenteric plexus (ICC-MP), interstitial cells of Cajal intramuscular (ICC-IM) and interstitial cells of Cajal deep muscular plexus (ICC-DMP) are the three types of ICC endowed within the intestinal muscle coat where they play different roles in gut motility. Studies on ICC ontogenesis showed ICC-MP in the human ileum by 7–9 weeks while information on ICC-IM and ICC-DMP in foetuses and new-borns are not exhaustive. Functional recordings in the fasting state of prematurely born babies aged 28–37 weeks showed immature ileal motility. To gain more information on the time of appearance of the three ICC types in the human ileum and on the steps of the acquisition of mature features, we studied by c-kit immuno-histochemistry foetuses aged 17–27 weeks and newborns aged 36–41 weeks. In parallel, the maturative steps of enteric plexuses and muscle layers were immunohistochemically examined by using anti-neuron specific enolase (NSE), anti-S-100 and anti-α smooth muscle actin (αSMA) antibodies. The appearance and differentiation of all the ICC types were seen to occur in concomitance with those of the related nerve plexuses and muscle layers. ICC-MP appeared first, ICC-IM and ICC-DMP later and their differentiation was incomplete at birth. In conclusion, the ICC-MP, the intestinal pacemaker cells, in spite of absence of food intake, are already present during the foetal life and the ICC-IM appear by pre-term life, thus ensuring neurotransmission. The ICC-DMP and their related nerve plexus and smooth muscle cells, i.e. the intestinal stretch receptor, begin to differentiate at birth. These findings might help in predicting neonatal ileal motor behaviour and in interpreting the role of ICC abnormalities in the pathophysiology of intestinal motile disorders of neonates and young children. PMID:17635640

  17. Expression of CYP3A4 and CYP3A7 in Human Foetal Tissues and its Correlation with Nuclear Receptors.

    PubMed

    Betts, Stina; Björkhem-Bergman, Linda; Rane, Anders; Ekström, Lena

    2015-10-01

    Previous reports have suggested that the nuclear receptors vitamin D receptor (VDR), peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are involved in the regulation of the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 expression in adults. The aim of this study was to investigate the gene expression of CYP3A4 and the foetal CYP3A7 in human foetal tissues and their relation to gene expression and genetic variations in the nuclear receptors VDR, PPARα, PXR and CAR. We determined the relative expression of CYP3A4 and CYP3A7 and these nuclear receptors in foetal livers, intestines and adrenals, using quantitative PCR. In addition, the expression of these enzymes was also analysed in adult liver. There was a high interindividual variability in CYP3A4 and CYP3A7, 49 times and 326 times, respectively. Both CYP3A4 and CYP3A7 had the highest expression in the liver. There were significant correlations (p < 0.001) between the nuclear receptors studied and the expression of CYP3A4 and CYP3A7 in foetal liver, as well as the expression of CYP3A4 in foetal intestine. Polymorphisms in the VDR gene, rs1544410 and rs1523130 (TaqI), in the PXR gene, rs1523130, and in the PPARα gene, rs4253728, were not correlated with CYP3A4 or CYP3A7 expression. However, C-homozygous individuals of the TaqI VDR polymorphism had 60% lower VDR gene expression (p < 0.05), than individuals carrying one or two T alleles. In conclusion, differences in the expression of nuclear receptors might determine the variability in CYP3A4 and CYP3A7 expression observed in foetal liver. PMID:25689036

  18. Mapping the Time Line of Development in Each Layer of Human Foetal Retina

    PubMed Central

    Kapoor, Kanchan; Sahni, Daisy; Singh, Balbir

    2016-01-01

    Introduction There is need to elucidate the histological developmental stages of various layers of retina, to understand the process better and provide clinically significant insights. Aim To study the details and chronology of in utero development of different layers of retina. Materials and Methods To study time of appearance, differentiation and organization of all layers of central retina in different gestational age groups. Retina was studied histologically in 27 foetuses from18th to 34th weeks of gestation. Results We found the period of mid gestation (19th -21st week of gestation) to be the defining time for retinal layers: The bruch’s membrane was fully formed at 20th week of gestation; the photoreceptor layer became well defined at 21st week of gestation; both the nuclear layers and both the plexiform layers and the ganglion cell layer became distinct at 19th week of gestation. Before the 19th week, outer and inner neuroblastic zones separated by the neuropil were seen. Well defined nerve fibre layer and inner limiting membrane was present at 18th week. The outer limiting membrane was first appreciated at 32nd week of gestation. Foetal retinal pigment epithelium was cuboidal and filled with melanin granules while no trace of lipofuscin pigment was found under fluorescent microscope. Conclusion Detailed data on retinal histogenesis and its timeline might aid in directed differentiation of retinal cell types from stem cells for therapeutic purposes. PMID:27134848

  19. Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system.

    PubMed

    Davis, Richard P; Nemes, Csilla; Varga, Eszter; Freund, Christian; Kosmidis, Georgios; Gkatzis, Konstantinos; de Jong, Danielle; Szuhai, Károly; Dinnyés, András; Mummery, Christine L

    2013-01-01

    Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application. PMID:23933400

  20. Characterisation of the human embryonic and foetal epicardium during heart development

    PubMed Central

    Risebro, Catherine A.; Vieira, Joaquim Miguel; Klotz, Linda; Riley, Paul R.

    2015-01-01

    The epicardium is essential for mammalian heart development. At present, our understanding of the timing and morphogenetic events leading to the formation of the human epicardium has essentially been extrapolated from model organisms. Here, we studied primary tissue samples to characterise human epicardium development. We reveal that the epicardium begins to envelop the myocardial surface at Carnegie stage (CS) 11 and this process is completed by CS15, earlier than previously inferred from avian studies. Contrary to prevailing dogma, the formed human epicardium is not a simple squamous epithelium and we reveal evidence of more complex structure, including novel spatial differences aligned to the developing chambers. Specifically, the ventricular, but not atrial, epicardium exhibited areas of expanded epithelium, preferential cell alignment and spindle-like morphology. Likewise, we reveal distinct properties ex vivo, such that ventricular cells spontaneously differentiate and lose epicardial identity, whereas atrial-derived cells remained ‘epithelial-like’. These data provide insight into the developing human epicardium that may contribute to our understanding of congenital heart disease and have implications for the development of strategies for endogenous cell-based cardiac repair. PMID:26395486

  1. Dimension of circumflex branch according to type of heart vascularisation in foetal life of human.

    PubMed

    Nowak, Dariusz; Boziłow, Władimir

    2003-02-01

    Typological differentiation of coronary arteries binds to various areas of vascularisation, which could suggest essential differences between vasculatory dimensions. There are not many papers analysing the influence of heart vascularisation on epicardial vessel dimensions during postnatal human life, furthermore, there are hardly any papers dealing with the prenatal period. The subject of examination was 188 human foetuses from 4th to 7th month of prenatal life. Foetuses were fixed for minimum 3 months in 9% formalin solution. They were taken from natural abortions and did not characterise any external malformations. There was a different number of foetuses in a variety of morphological age groups. Adachi classification was used to describe all types: type I--classical, with equal coronary arteries, type II--predominance of right coronary artery, type III--predominance of left coronary artery. Speed of circumflex branch growth in different types of vascularisation is various. Analysis of the differences among dimensions of artery in various types showed there are statistically crucial ones, especially between: types III and I or types III and II. PMID:12769180

  2. Advances in Human B Cell Phenotypic Profiling

    PubMed Central

    Kaminski, Denise A.; Wei, Chungwen; Qian, Yu; Rosenberg, Alexander F.; Sanz, Ignacio

    2012-01-01

    To advance our understanding and treatment of disease, research immunologists have been called-upon to place more centralized emphasis on impactful human studies. Such endeavors will inevitably require large-scale study execution and data management regulation (“Big Biology”), necessitating standardized and reliable metrics of immune status and function. A well-known example setting this large-scale effort in-motion is identifying correlations between eventual disease outcome and T lymphocyte phenotype in large HIV-patient cohorts using multiparameter flow cytometry. However, infection, immunodeficiency, and autoimmunity are also characterized by correlative and functional contributions of B lymphocytes, which to-date have received much less attention in the human Big Biology enterprise. Here, we review progress in human B cell phenotyping, analysis, and bioinformatics tools that constitute valuable resources for the B cell research community to effectively join in this effort. PMID:23087687

  3. Isolation of cardiac myosin light-chain isotypes by chromatofocusing. Comparison of human cardiac atrial light-chain 1 and foetal ventricular light-chain 1.

    PubMed

    Vincent, N D; Cummins, P

    1985-04-01

    Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and

  4. The impact of trisomy 21 on foetal haematopoiesis

    PubMed Central

    Roberts, Irene; O'Connor, David; Roy, Anindita; Cowan, Gillian; Vyas, Paresh

    2015-01-01

    The high frequency of a unique neonatal preleukaemic syndrome, Transient Abnormal Myelopoiesis (TAM), and subsequent acute myeloid leukaemia in early childhood in patients with trisomy 21 (Down syndrome) points to a specific role for trisomy 21 in transforming foetal haematopoietic cells. N-terminal truncating mutations in the key haematopoietic transcription factor GATA1 are acquired during foetal life in virtually every case. These mutations are not leukaemogenic in the absence of trisomy 21. In mouse models, deregulated expression of chromosome 21-encoded genes is implicated in leukaemic transformation, but does not recapitulate the effects of trisomy 21 in a human context. Recent work using primary human foetal liver and bone marrow cells, human embryonic stem cells and iPS cells cells shows that prior to acquistion of GATA1 mutations, trisomy 21 itself alters human foetal haematopoietic stem cell and progenitor cell biology causing multiple abnormalities in myelopoiesis and B-lymphopoiesis. The molecular basis by which trisomy 21 exerts these effects is likely to be extremely complex, to be tissue- and lineage-specific and to be dependent on ontogeny-related characteristics of the foetal microenvironment. PMID:23932236

  5. Decomposing Phenotype Descriptions for the Human Skeletal Phenome

    PubMed Central

    Groza, Tudor; Hunter, Jane; Zankl, Andreas

    2013-01-01

    Over the course of the last few years there has been a significant amount of research performed on ontology-based formalization of phenotype descriptions. The intrinsic value and knowledge captured within such descriptions can only be expressed by taking advantage of their inner structure that implicitly combines qualities and anatomical entities. We present a meta-model (the Phenotype Fragment Ontology) and a processing pipeline that enable together the automatic decomposition and conceptualization of phenotype descriptions for the human skeletal phenome. We use this approach to showcase the usefulness of the generic concept of phenotype decomposition by performing an experimental study on all skeletal phenotype concepts defined in the Human Phenotype Ontology. PMID:23440304

  6. The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data.

    PubMed

    Köhler, Sebastian; Doelken, Sandra C; Mungall, Christopher J; Bauer, Sebastian; Firth, Helen V; Bailleul-Forestier, Isabelle; Black, Graeme C M; Brown, Danielle L; Brudno, Michael; Campbell, Jennifer; FitzPatrick, David R; Eppig, Janan T; Jackson, Andrew P; Freson, Kathleen; Girdea, Marta; Helbig, Ingo; Hurst, Jane A; Jähn, Johanna; Jackson, Laird G; Kelly, Anne M; Ledbetter, David H; Mansour, Sahar; Martin, Christa L; Moss, Celia; Mumford, Andrew; Ouwehand, Willem H; Park, Soo-Mi; Riggs, Erin Rooney; Scott, Richard H; Sisodiya, Sanjay; Van Vooren, Steven; Wapner, Ronald J; Wilkie, Andrew O M; Wright, Caroline F; Vulto-van Silfhout, Anneke T; de Leeuw, Nicole; de Vries, Bert B A; Washingthon, Nicole L; Smith, Cynthia L; Westerfield, Monte; Schofield, Paul; Ruef, Barbara J; Gkoutos, Georgios V; Haendel, Melissa; Smedley, Damian; Lewis, Suzanna E; Robinson, Peter N

    2014-01-01

    The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online. PMID:24217912

  7. The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data

    PubMed Central

    Köhler, Sebastian; Doelken, Sandra C.; Mungall, Christopher J.; Bauer, Sebastian; Firth, Helen V.; Bailleul-Forestier, Isabelle; Black, Graeme C. M.; Brown, Danielle L.; Brudno, Michael; Campbell, Jennifer; FitzPatrick, David R.; Eppig, Janan T.; Jackson, Andrew P.; Freson, Kathleen; Girdea, Marta; Helbig, Ingo; Hurst, Jane A.; Jähn, Johanna; Jackson, Laird G.; Kelly, Anne M.; Ledbetter, David H.; Mansour, Sahar; Martin, Christa L.; Moss, Celia; Mumford, Andrew; Ouwehand, Willem H.; Park, Soo-Mi; Riggs, Erin Rooney; Scott, Richard H.; Sisodiya, Sanjay; Vooren, Steven Van; Wapner, Ronald J.; Wilkie, Andrew O. M.; Wright, Caroline F.; Vulto-van Silfhout, Anneke T.; de Leeuw, Nicole; de Vries, Bert B. A.; Washingthon, Nicole L.; Smith, Cynthia L.; Westerfield, Monte; Schofield, Paul; Ruef, Barbara J.; Gkoutos, Georgios V.; Haendel, Melissa; Smedley, Damian; Lewis, Suzanna E.; Robinson, Peter N.

    2014-01-01

    The Human Phenotype Ontology (HPO) project, available at http://www.human-phenotype-ontology.org, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online. PMID:24217912

  8. Foetal Testosterone, Social Relationships, and Restricted Interests in Children

    ERIC Educational Resources Information Center

    Knickmeyer, Rebecca; Baron-Cohen, Simon; Raggatt, Peter; Taylor, Kevin

    2005-01-01

    Background: Sex-differences exist in some areas of human social behaviour. In animals, foetal testosterone (fT) plays a central role in organising the brain and in later social behaviour. fT has also been implicated in language development, eye-contact, and spatial ability in humans. Methods: Fifty-eight children (35 male and 23 female), whose fT…

  9. Olfactory phenotypic expression unveils human aging

    PubMed Central

    Mazzatenta, Andrea; Cellerino, Alessandro; Origlia, Nicola; Barloscio, Davide; Sartucci, Ferdinando; Giulio, Camillo Di; Domenici, Luciano

    2016-01-01

    The mechanism of the natural aging of olfaction and its declinein the absence of any overt disease conditions remains unclear. Here, we investigated this mechanism through measurement of one of the parameters of olfactory function, the absolute threshold, in a healthy population from childhood to old age. The absolute olfactory threshold data were collected from an Italian observational study with 622 participants aged 5-105 years. A subjective testing procedure of constant stimuli was used, which was also compared to the ‘staircase’ method, with the calculation of the reliability. The n-butanol stimulus was used as an ascending series of nine molar concentrations that were monitored using an electronic nose. The data were analyzed using nonparametric statistics because of the multimodal distribution. We show that the age-related variations in the absolute olfactory threshold are not continuous; instead, there are multiple olfactory phenotypes. Three distinct age-related phenotypes were defined, termed as ‘juvenile’, ‘mature’ and ‘elder’. The frequency of these three phenotypes depends on age. Our data suggest that the sense of smell does not decrease linearly with aging. Our findings provide the basis for further understanding of olfactory loss as an anticipatory sign of aging and neurodegenerative processes. PMID:27027240

  10. The phenotypic legacy of admixture between modern humans and Neandertals.

    PubMed

    Simonti, Corinne N; Vernot, Benjamin; Bastarache, Lisa; Bottinger, Erwin; Carrell, David S; Chisholm, Rex L; Crosslin, David R; Hebbring, Scott J; Jarvik, Gail P; Kullo, Iftikhar J; Li, Rongling; Pathak, Jyotishman; Ritchie, Marylyn D; Roden, Dan M; Verma, Shefali S; Tromp, Gerard; Prato, Jeffrey D; Bush, William S; Akey, Joshua M; Denny, Joshua C; Capra, John A

    2016-02-12

    Many modern human genomes retain DNA inherited from interbreeding with archaic hominins, such as Neandertals, yet the influence of this admixture on human traits is largely unknown. We analyzed the contribution of common Neandertal variants to over 1000 electronic health record (EHR)-derived phenotypes in ~28,000 adults of European ancestry. We discovered and replicated associations of Neandertal alleles with neurological, psychiatric, immunological, and dermatological phenotypes. Neandertal alleles together explained a significant fraction of the variation in risk for depression and skin lesions resulting from sun exposure (actinic keratosis), and individual Neandertal alleles were significantly associated with specific human phenotypes, including hypercoagulation and tobacco use. Our results establish that archaic admixture influences disease risk in modern humans, provide hypotheses about the effects of hundreds of Neandertal haplotypes, and demonstrate the utility of EHR data in evolutionary analyses. PMID:26912863

  11. The phenotypic legacy of admixture between modern humans and Neanderthals

    PubMed Central

    Simonti, Corinne N.; Vernot, Benjamin; Bastarache, Lisa; Bottinger, Erwin; Carrell, David S.; Chisholm, Rex L.; Crosslin, David R.; Hebbring, Scott J.; Jarvik, Gail P.; Kullo, Iftikhar J.; Li, Rongling; Pathak, Jyotishman; Ritchie, Marylyn D.; Roden, Dan M.; Verma, Shefali S.; Tromp, Gerard; Prato, Jeffrey D.; Bush, William S.; Akey, Joshua M.; Denny, Joshua C.; Capra, John A.

    2016-01-01

    Many modern human genomes retain DNA inherited from interbreeding with archaic hominins, such as Neanderthals, yet the influence of this admixture on human traits is largely unknown. We analyzed the contribution of common Neanderthal variants to over 1,000 electronic health record (EHR)-derived phenotypes in ~28,000 adults of European ancestry. We discovered and replicated associations of Neanderthal alleles with neurological, psychiatric, immunological, and dermatological phenotypes. Neanderthal alleles together explain a significant fraction of the variation in risk for depression and skin lesions resulting from sun exposure (actinic keratosis), and individual Neanderthal alleles are significantly associated with specific human phenotypes, including hypercoagulation and tobacco use. Our results establish that archaic admixture influences disease risk in modern humans, provide hypotheses about the effects of hundreds of Neanderthal haplotypes and demonstrate the utility of EHR data in evolutionary analyses. PMID:26912863

  12. Foetal therapy: what works? Closed interatrial septum.

    PubMed

    Herberg, Ulrike; Berg, Christoph; Geipel, Annegret; Gembruch, Ulrich; Breuer, Johannes

    2014-10-01

    Hypoplastic left-heart syndrome and critical aortic stenosis with severely restricted or intact foramen ovale are associated with high neonatal mortality and poor long-term outcome. Despite accurate foetal diagnosis and successful postnatal catheter-based and surgical intervention, the 1-month survival rate is about 33%. Changes in pulmonary vascular architecture resulting in pulmonary hypertension result in important long-term morbidity. Prenatal relief of left atrial and pulmonary hypertension may promote normal pulmonary vascular and parenchymal development and improve short- and long-term outcomes. Foetal atrial balloon septostomy, laser perforation, and stenting of the foetal interatrial septum are the current options for foetal therapy. This paper provides an overview of foetal diagnosis, selection of patients for foetal intervention, and interventional techniques, and also reviews the current status of foetal and postnatal outcomes after intrauterine intervention. PMID:25159557

  13. Loss of gene function and evolution of human phenotypes

    PubMed Central

    Oh, Hye Ji; Choi, Dongjin; Goh, Chul Jun; Hahn, Yoonsoo

    2015-01-01

    Humans have acquired many distinct evolutionary traits after the human-chimpanzee divergence. These phenotypes have resulted from genetic changes that occurred in the human genome and were retained by natural selection. Comparative primate genome analyses reveal that loss-of-function mutations are common in the human genome. Some of these gene inactivation events were revealed to be associated with the emergence of advantageous phenotypes and were therefore positively selected and fixed in modern humans (the “less-ismore” hypothesis). Representative cases of human gene inactivation and their functional implications are presented in this review. Functional studies of additional inactive genes will provide insight into the molecular mechanisms underlying acquisition of various human-specific traits. [BMB Reports 2015; 48(7): 373-379] PMID:25887751

  14. The gray phenotype and tristable phenotypic transitions in the human fungal pathogen Candida tropicalis.

    PubMed

    Zhang, Yulong; Tao, Li; Zhang, Qiuyu; Guan, Guobo; Nobile, Clarissa J; Zheng, Qiushi; Ding, Xuefen; Huang, Guanghua

    2016-08-01

    Phenotypic plasticity, the ability to switch between different morphological types, plays critical roles in environmental adaptation, leading to infections, and allowing for sexual reproduction in pathogenic Candida species. Candida tropicalis, which is both an emerging human fungal pathogen and an environmental fungus, can switch between two heritable cell types termed white and opaque. In this study, we report the discovery of a novel phenotype in C. tropicalis, named the gray phenotype. Similar to Candida albicans and Candida dubliniensis, white, gray, and opaque cell types of C. tropicalis also form a tristable switching system, where gray cells are relatively small and elongated. In C. tropicalis, gray cells exhibit intermediate levels of mating competency and virulence in a mouse systemic infection model compared to the white and opaque cell types, express a set of cell type-enriched genes, and exhibit both common and species-specific biological features. The key regulators of white-opaque transitions, Wor1 and Efg1, are not required for the gray phenotype. A comparative study of the gray phenotypes in C. tropicalis, C. albicans, and C. dubliniensis provides clues to explain the virulence properties and niche preferences of C. tropicalis. PMID:27246518

  15. Systematic discovery of nonobvious human disease models through orthologous phenotypes.

    PubMed

    McGary, Kriston L; Park, Tae Joo; Woods, John O; Cha, Hye Ji; Wallingford, John B; Marcotte, Edward M

    2010-04-01

    Biologists have long used model organisms to study human diseases, particularly when the model bears a close resemblance to the disease. We present a method that quantitatively and systematically identifies nonobvious equivalences between mutant phenotypes in different species, based on overlapping sets of orthologous genes from human, mouse, yeast, worm, and plant (212,542 gene-phenotype associations). These orthologous phenotypes, or phenologs, predict unique genes associated with diseases. Our method suggests a yeast model for angiogenesis defects, a worm model for breast cancer, mouse models of autism, and a plant model for the neural crest defects associated with Waardenburg syndrome, among others. Using these models, we show that SOX13 regulates angiogenesis, and that SEC23IP is a likely Waardenburg gene. Phenologs reveal functionally coherent, evolutionarily conserved gene networks-many predating the plant-animal divergence-capable of identifying candidate disease genes. PMID:20308572

  16. RNA Directed Modulation of Phenotypic Plasticity in Human Cells

    PubMed Central

    Burdach, Jon; Morris, Kevin V.

    2016-01-01

    Natural selective processes have been known to drive phenotypic plasticity, which is the emergence of different phenotypes from one genome following environmental stimulation. Long non-coding RNAs (lncRNAs) have been observed to modulate transcriptional and epigenetic states of genes in human cells. We surmised that lncRNAs are governors of phenotypic plasticity and drive natural selective processes through epigenetic modulation of gene expression. Using heat shocked human cells as a model we find several differentially expressed transcripts with the top candidates being lncRNAs derived from retro-elements. One particular retro-element derived transcripts, Retro-EIF2S2, was found to be abundantly over-expressed in heat shocked cells. Over-expression of Retro-EIF2S2 significantly enhanced cell viability and modulated a predisposition for an adherent cellular phenotype upon heat shock. Mechanistically, we find that this retro-element derived transcript interacts directly with a network of proteins including 40S ribosomal protein S30 (FAU), Eukaryotic translation initiation factor 5A (EIF5A), and Ubiquitin-60S ribosomal protein L40 (UBA52) to affect protein modulated cell adhesion pathways. We find one motif in Retro-EIF2S2 that exhibits binding to FAU and modulates phenotypic cell transitions from adherent to suspension states. The observations presented here suggest that retroviral derived transcripts actively modulate phenotypic plasticity in human cells in response to environmental selective pressures and suggest that natural selection may play out through the action of retro-elements in human cells. PMID:27082860

  17. RNA Directed Modulation of Phenotypic Plasticity in Human Cells.

    PubMed

    Trakman, Laura; Hewson, Chris; Burdach, Jon; Morris, Kevin V

    2016-01-01

    Natural selective processes have been known to drive phenotypic plasticity, which is the emergence of different phenotypes from one genome following environmental stimulation. Long non-coding RNAs (lncRNAs) have been observed to modulate transcriptional and epigenetic states of genes in human cells. We surmised that lncRNAs are governors of phenotypic plasticity and drive natural selective processes through epigenetic modulation of gene expression. Using heat shocked human cells as a model we find several differentially expressed transcripts with the top candidates being lncRNAs derived from retro-elements. One particular retro-element derived transcripts, Retro-EIF2S2, was found to be abundantly over-expressed in heat shocked cells. Over-expression of Retro-EIF2S2 significantly enhanced cell viability and modulated a predisposition for an adherent cellular phenotype upon heat shock. Mechanistically, we find that this retro-element derived transcript interacts directly with a network of proteins including 40S ribosomal protein S30 (FAU), Eukaryotic translation initiation factor 5A (EIF5A), and Ubiquitin-60S ribosomal protein L40 (UBA52) to affect protein modulated cell adhesion pathways. We find one motif in Retro-EIF2S2 that exhibits binding to FAU and modulates phenotypic cell transitions from adherent to suspension states. The observations presented here suggest that retroviral derived transcripts actively modulate phenotypic plasticity in human cells in response to environmental selective pressures and suggest that natural selection may play out through the action of retro-elements in human cells. PMID:27082860

  18. Phenotypic variability in human skin mast cells.

    PubMed

    Babina, Magda; Guhl, Sven; Artuc, Metin; Trivedi, Neil N; Zuberbier, Torsten

    2016-06-01

    Mast cells (MCs) are unique constituents of the human body. While inter-individual differences may influence the ways by which MCs operate in their skin habitat, they have not been surveyed in a comprehensive manner so far. We therefore set out to quantify skin MC variability in a large cohort of subjects. Pathophysiologically relevant key features were quantified and correlated: transcripts of c-kit, FcεRIα, FcεRIβ, FcεRIγ, histidine decarboxylase, tryptase, and chymase; surface expression of c-Kit, FcεRIα; activity of tryptase, and chymase; histamine content and release triggered by FcεRI and Ca(2+) ionophore. While there was substantial variability among subjects, it strongly depended on the feature under study (coefficient of variation 33-386%). Surface expression of FcεRI was positively associated with FcεRIα mRNA content, histamine content with HDC mRNA, and chymase activity with chymase mRNA. Also, MC signature genes were co-regulated in distinct patterns. Intriguingly, histamine levels were positively linked to tryptase and chymase activity, whereas tryptase and chymase activity appeared to be uncorrelated. FcεRI triggered histamine release was highly variable and was unrelated to FcεRI expression but unexpectedly tightly correlated with histamine release elicited by Ca(2+) ionophore. This most comprehensive and systematic work of its kind provides not only detailed insights into inter-individual variability in MCs, but also uncovers unexpected patterns of co-regulation among signature attributes of the lineage. Differences in MCs among humans may well underlie clinical responses in settings of allergic reactions and complex skin disorders alike. PMID:26706922

  19. Mouse genetic and phenotypic resources for human genetics

    PubMed Central

    Schofield, Paul N.; Hoehndorf, Robert; Gkoutos, Georgios V.

    2012-01-01

    The use of model organisms to provide information on gene function has proved to be a powerful approach to our understanding of both human disease and fundamental mammalian biology. Large-scale community projects using mice, based on forward and reverse genetics, and now the pan-genomic phenotyping efforts of the International Mouse Phenotyping Consortium (IMPC), are generating resources on an unprecedented scale which will be extremely valuable to human genetics and medicine. We discuss the nature and availability of data, mice and ES cells from these large-scale programmes, the use of these resources to help prioritise and validate candidate genes in human genetic association studies, and how they can improve our understanding of the underlying pathobiology of human disease. PMID:22422677

  20. Improved human disease candidate gene prioritization using mouse phenotype

    PubMed Central

    Chen, Jing; Xu, Huan; Aronow, Bruce J; Jegga, Anil G

    2007-01-01

    Background The majority of common diseases are multi-factorial and modified by genetically and mechanistically complex polygenic interactions and environmental factors. High-throughput genome-wide studies like linkage analysis and gene expression profiling, tend to be most useful for classification and characterization but do not provide sufficient information to identify or prioritize specific disease causal genes. Results Extending on an earlier hypothesis that the majority of genes that impact or cause disease share membership in any of several functional relationships we, for the first time, show the utility of mouse phenotype data in human disease gene prioritization. We study the effect of different data integration methods, and based on the validation studies, we show that our approach, ToppGene , outperforms two of the existing candidate gene prioritization methods, SUSPECTS and ENDEAVOUR. Conclusion The incorporation of phenotype information for mouse orthologs of human genes greatly improves the human disease candidate gene analysis and prioritization. PMID:17939863

  1. Structural Modeling Insights into Human VKORC1 Phenotypes

    PubMed Central

    Czogalla, Katrin J.; Watzka, Matthias; Oldenburg, Johannes

    2015-01-01

    Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) catalyses the reduction of vitamin K and its 2,3-epoxide essential to sustain γ-carboxylation of vitamin K-dependent proteins. Two different phenotypes are associated with mutations in human VKORC1. The majority of mutations cause resistance to 4-hydroxycoumarin- and indandione-based vitamin K antagonists (VKA) used in the prevention and therapy of thromboembolism. Patients with these mutations require greater doses of VKA for stable anticoagulation than patients without mutations. The second phenotype, a very rare autosomal-recessive bleeding disorder caused by combined deficiency of vitamin K dependent clotting factors type 2 (VKCFD2) arises from a homozygous Arg98Trp mutation. The bleeding phenotype can be corrected by vitamin K administration. Here, we summarize published experimental data and in silico modeling results in order to rationalize the mechanisms of VKA resistance and VKCFD2. PMID:26287237

  2. The DDBJ Japanese Genotype-phenotype Archive for genetic and phenotypic human data

    PubMed Central

    Kodama, Yuichi; Mashima, Jun; Kosuge, Takehide; Katayama, Toshiaki; Fujisawa, Takatomo; Kaminuma, Eli; Ogasawara, Osamu; Okubo, Kousaku; Takagi, Toshihisa; Nakamura, Yasukazu

    2015-01-01

    The DNA Data Bank of Japan Center (DDBJ Center; http://www.ddbj.nig.ac.jp) maintains and provides public archival, retrieval and analytical services for biological information. Since October 2013, DDBJ Center has operated the Japanese Genotype-phenotype Archive (JGA) in collaboration with our partner institute, the National Bioscience Database Center (NBDC) of the Japan Science and Technology Agency. DDBJ Center provides the JGA database system which securely stores genotype and phenotype data collected from individuals whose consent agreements authorize data release only for specific research use. NBDC has established guidelines and policies for sharing human-derived data and reviews data submission and usage requests from researchers. In addition to the JGA project, DDBJ Center develops Semantic Web technologies for data integration and sharing in collaboration with the Database Center for Life Science. This paper describes the overview of the JGA project, updates to the DDBJ databases, and services for data retrieval, analysis and integration. PMID:25477381

  3. Does human ejaculate quality relate to phenotypic traits?

    PubMed

    Jeffery, Austin John; Pham, Michael N; Shackelford, Todd K; Fink, Bernhard

    2016-05-01

    A given man's phenotype embodies cues of his ancestral ability to effectively defend himself and his kin from harm, to survive adverse conditions, and to acquire status and mating opportunities. In this review, we explore the hypothesis that a man's phenotype also embodies cues to fertility or the probability that an ejaculate will fertilize ova. Female mate choice depends on the ability to discern the quality of a male reproductive partner through his phenotype, and male fertility may be among the traits that females have evolved to detect. A female who selects as mates males that deliver higher quality ejaculates will, on average, be more fecund than her competitors. Data on several non-human species demonstrate correlations between ejaculate quality and secondary sexual characteristics that inform female mate choice, suggesting that females may select mates in part on the basis of fertility. While the non-human literature on this topic has advanced, the human literature remains limited in scope and there is no clear consensus on appropriate methodologies or theoretical positions. We provide a comprehensive review and meta-analysis of this literature, and conclude by proposing solutions to the many issues that impede progress in the field. In the process, we hope to encourage interest and insight from investigators in other areas of human mating and reproductive biology. Am. J. Hum. Biol. 28:318-329, 2016. © 2015 Wiley Periodicals, Inc. PMID:26626022

  4. Foetal and neonatal alloimmune thrombocytopaenia

    PubMed Central

    Kaplan, Cecile

    2006-01-01

    Foetal/neonatal alloimmune thrombocytopaenia (NAIT) results from maternal alloimmunisation against foetal platelet antigens inherited from the father and different from those present in the mother, and usually presents as a severe isolated thrombocytopaenia in otherwise healthy newborns. The incidence has been estimated at 1/800 to 1/1 000 live births. NAIT has been considered to be the platelet counterpart of Rh Haemolytic Disease of the Newborn (RHD). Unlike RHD, NAIT can occur during a first pregnancy. The spectrum of the disease may range from sub-clinical moderate thrombocytopaenia to life-threatening bleeding in the neonatal period. Mildly affected infants may be asymptomatic. In those with severe thrombocytopaenia, the most common presentations are petechiae, purpura or cephalohaematoma at birth, associated with major risk of intracranial haemorrhage (up to 20% of reported cases), which leads to death or neurological sequelae. Alloimmune thrombocytopaenia is more often unexpected and is usually diagnosed after birth. Once suspected, the diagnosis is confirmed by demonstration of maternal antiplatelet alloantibodies directed against a paternal antigen inherited by the foetus/neonate. Post-natal management involves transfusion of platelets devoid of this antigen, and should not be delayed by biological confirmation of the diagnosis (once the diagnosis is suspected), especially in case of severe thrombocytopaenia. Prompt diagnosis and treatment are essential to reduce the chances of death and disability due to haemorrhage. Due to the high rate of recurrence and increased severity of the foetal thrombocytopaenia in successive pregnancies, antenatal therapy should be offered. However, management of high-risk pregnancies is still a matter of discussion. PMID:17032445

  5. Human Cancers Express a Mutator Phenotype: Hypothesis, Origin, and Consequences

    PubMed Central

    Loeb, Lawrence A.

    2016-01-01

    The mutator phenotype hypothesis was postulated more than 40 years ago. It was based on the multiple enzymatic steps required to precisely replicate the 6 billion bases in the human genome each time a normal cell divides. A reduction in this accuracy during tumor progression could be responsible for the striking heterogeneity of malignant cells within a tumor and for the rapidity by which cancers become resistant to therapy. PMID:27197248

  6. Do Thyroid Disrupting Chemicals Influence Foetal Development during Pregnancy?

    PubMed Central

    Hartoft-Nielsen, Marie-Louise; Boas, Malene; Bliddal, Sofie; Rasmussen, Åase Krogh; Main, Katharina; Feldt-Rasmussen, Ulla

    2011-01-01

    Maternal euthyroidism during pregnancy is crucial for normal development and, in particular, neurodevelopment of the foetus. Up to 3.5 percent of pregnant women suffer from hypothyroidism. Industrial use of various chemicals—endocrine disrupting chemicals (EDCs)—has been shown to cause almost constant exposure of humans with possible harmful influence on health and hormone regulation. EDCs may affect thyroid hormone homeostasis by different mechanisms, and though the effect of each chemical seems scarce, the added effects may cause inappropriate consequences on, for example, foetal neurodevelopment. This paper focuses on thyroid hormone influence on foetal development in relation to the chemicals suspected of thyroid disrupting properties with possible interactions with maternal thyroid homeostasis. Knowledge of the effects is expected to impact the general debate on the use of these chemicals. However, more studies are needed to elucidate the issue, since human studies are scarce. PMID:21918727

  7. Our bodies, our choices: Australian law on foetal personhood.

    PubMed

    Anolak, Helena

    2015-03-01

    The debate revolving around foetal vs maternal rights has been philosophised for years. It has captured attention internationally with the European Court of Human Rights struggling for over five decades to define personhood, and is still yet to reach a definite formulation. A proposed Law Crimes Amendment Bill (Zoe's Law, 2) is currently fuelling public debate about women's reproductive choices in New South Wales, Australia. The proposed legal bill attempts to redefine a 'person' or 'human being' by placing a marker on when 'personhood begins' namely at 20 weeks or weighing a minimum of 400g. Similar laws recognising personhood at foetal viability have come into force in the United States of America that clearly show the broader consequences of this kind of legislation as American women now face county-by-county, state-by-state anti-choice legislative activism. Midwives work closely with women and their families giving them the authority to formulate opinions on issues of maternal-foetal conflict. If a law such as this is allowed to pass a number of legal and ethical issues will arise for Australian midwives that could potentially have far reaching implications for them and for the women and families that they provide women-centred care for. PMID:25458964

  8. Human cytotrophoblasts acquire aneuploidies as they differentiateto an invasive phenotype

    SciTech Connect

    Weier, Jingly F.; Weier, Heinz-Ulrich G.; Jung, Christine J.; Gormley, Matthew; Zhou, Yuan; Chu, Lisa W.; Genbacev, Olga; Wright, AlexiA.; Fisher, Susan J.

    2004-12-15

    Through an unusual differentiation process, human trophoblast progenitors (cytotrophoblasts) give rise to tumor-like cells that invade the uterus. By an unknown mechanism, invasive cytotrophoblasts exhibit permanent cell cycle withdrawal. Here we report molecular cytogenetic data showing that {approx} 20 to 60 percent of these interphase cells had acquired aneusomies involving chromosomes X, Y, o r16. The incidence positively correlated with gestational age and differentiation to an invasive phenotype. Scoring 12 chromosomes in flow-sorted cytotrophoblasts showed that more than 95 percent of the cells were hyperdiploid. Thus, aneuploidy appears to be an important component of normal placentation, perhaps limiting the proliferative and invasive potential of cytotrophoblasts within the uterus.

  9. Genetic and phenotypic consequences of introgression between humans and Neanderthals.

    PubMed

    Wills, Christopher

    2011-01-01

    Strong evidence for introgression of Neanderthal genes into parts of the modern human gene pool has recently emerged. The evidence indicates that some populations of modern humans have received infusions of genes from two different groups of Neanderthals. One of these Neanderthal groups lived in the Middle East and Central Europe and the other group (the Denisovans) is known to have lived in Central Asia and was probably more widespread. This review examines two questions. First, how were these introgressions detected and what does the genetic evidence tell us about their nature and extent? We will see that an unknown but possibly large fraction of the entire Neanderthal gene complement may have survived in modern humans. Even though each modern European and Asian carries only a few percent of genes that can be traced back to Neanderthals, different individuals carry different subgroups of these introgressed genes. Second, what is the likelihood that this Neanderthal genetic legacy has had phenotypic effects on modern humans? We examine evidence for and against the possibility that some of the surviving fragments of Neanderthal genomes have been preserved by natural selection, and we explore the ways in which more evidence bearing on this question will become available in the future. PMID:22099691

  10. Three electrophysiological phenotypes of cultured human umbilical vein endothelial cells.

    PubMed

    Yu, K; Ruan, D Y; Ge, S Y

    2002-09-01

    The conventional whole cell patch-clamp technique was used to measure the resting membrane conductance and membrane currents of nonstimulated cultured human umbilical vein endothelial cells (HUVECs) in different ionic conditions. Three electrophysiological phenotypes of cultured HUVECs (n = 122) were determined: first, 20% of cells as type I mainly displaying the inwardly rectifying potassium current (IKi); second, 38% of cells as type II in which IKi was super-posed on a TEA-sensitive, delayed rectifying current; third, 27% of cells as type III predominantly displaying the outwardly rectifying current which was sensitive to TEA and slightly inhibited by a chloride channel blocker niflumic acid (N.A.). In cells of type I, the mean zero-current potential (V0) was dependent on extracellular K+ ([K+]o) but not on Cl-, indicating major permeability to K+. Whereas V0 of type II was also affected by extracellular Cl- ([Cl-]o), indicating the contribution of an outward Cl- current in setting V0. The cells of type III were not sensitive to decrease of [Cl-]o and the outward current was activated in a relative stable voltage range. This varying phenotypic expression and multipotential behavior of HUVECs suggests that the electrical features of HUVEC may be primarily determined by embryonic origin and local effect of the microenvironment. This research provided the detailed electrophysiological knowledge of the endothelial cells. PMID:12537354

  11. Estimation of foetal brain dose from I-131 in the foetal thyroid

    NASA Astrophysics Data System (ADS)

    O'Hare, N. J.; Gilligan, P.; Murphy, D.; Malone, J. F.

    1997-09-01

    The ingestion of I-131 by pregnant women can have consequences for the developing foetus, in particular brain function. As the foetal thyroid accumulates iodine from the twelfth week of gestation onwards, the determination of foetal brain dose resulting from such I-131 accumulation is essential. Normal dosimetric methods fail to treat the case of the foetus. Using an approximation method based on the MIRD approach, a foetal dose estimation scheme is developed to allow the determination of foetal brain dose from foetal thyroid irradiation. Dose values are obtained for the foetus based on the maternal intake of I-131. It was found that the choice of biokinetic model for the mother/foetus has a large impact on the determined dose estimate.

  12. The Evolution of Human Genetic and Phenotypic Variation in Africa

    PubMed Central

    Campbell, Michael C.

    2010-01-01

    Africa is the birthplace of modern humans, and is the source of the geographic expansion of ancestral populations into other regions of the world. Indigenous Africans are characterized by high levels of genetic diversity within and between populations. The pattern of genetic variation in these populations has been shaped by demographic events occurring over the last 200,000 years. The dramatic variation in climate, diet, and exposure to infectious disease across the continent has also resulted in novel genetic and phenotypic adaptations in extant Africans. This review summarizes some recent advances in our understanding of the demographic history and selective pressures that have influenced levels and patterns of diversity in African populations. PMID:20178763

  13. Reconstructing phylogenies and phenotypes: a molecular view of human evolution.

    PubMed

    Bradley, Brenda J

    2008-04-01

    This review broadly summarizes how molecular biology has contributed to our understanding of human evolution. Molecular anthropology began in the 1960s with immunological comparisons indicating that African apes and humans were closely related and, indeed, shared a common ancestor as recently as 5 million years ago. Although initially dismissed, this finding has proven robust and numerous lines of molecular evidence now firmly place the human-ape divergence at 4-8 Ma. Resolving the trichotomy among humans, chimpanzees and gorillas took a few more decades. Despite the readily apparent physical similarities shared by African apes to the exclusion of modern humans (body hair, knuckle-walking, thin tooth enamel), the molecular support for a human-chimpanzee clade is now overwhelming. More recently, whole genome sequencing and gene mapping have shifted the focus of molecular anthropology from phylogenetic analyses to phenotypic reconstruction and functional genomics. We are starting to identify the genetic basis of the morphological, physiological and behavioural traits that distinguish modern humans from apes and apes from other primates. Most notably, recent comparative genomic analyses strongly indicate that the marked differences between modern humans and chimpanzees are likely due more to changes in gene regulation than to modifications of the genes themselves, an idea first proposed over 30 years ago. Almost weekly, press releases describe newly identified genes and regulatory elements that seem to have undergone strong positive selection along the human lineage. Loci involved in speech (e.g. FOXP2), brain development (e.g. ASPM), and skull musculature (e.g. MYH16) have been of particular interest, but some surprising candidate loci (e.g. those involved in auditory capabilities) have emerged as well. Exciting new research avenues, such as the Neanderthal Genome Project, promise that molecular analyses will continue to provide novel insights about our evolution

  14. Dissecting phenotypic traits linked to human resilience to Alzheimer's pathology.

    PubMed

    Perez-Nievas, Beatriz G; Stein, Thor D; Tai, Hwan-Ching; Dols-Icardo, Oriol; Scotton, Thomas C; Barroeta-Espar, Isabel; Fernandez-Carballo, Leticia; de Munain, Estibaliz Lopez; Perez, Jesus; Marquie, Marta; Serrano-Pozo, Alberto; Frosch, Mathew P; Lowe, Val; Parisi, Joseph E; Petersen, Ronald C; Ikonomovic, Milos D; López, Oscar L; Klunk, William; Hyman, Bradley T; Gómez-Isla, Teresa

    2013-08-01

    Clinico-pathological correlation studies and positron emission tomography amyloid imaging studies have shown that some individuals can tolerate substantial amounts of Alzheimer's pathology in their brains without experiencing dementia. Few details are known about the neuropathological phenotype of these unique cases that might prove relevant to understanding human resilience to Alzheimer's pathology. We conducted detailed quantitative histopathological and biochemical assessments on brains from non-demented individuals before death whose brains were free of substantial Alzheimer's pathology, non-demented individuals before death but whose post-mortem examination demonstrated significant amounts of Alzheimer's changes ('mismatches'), and demented Alzheimer's cases. Quantification of amyloid-β plaque burden, stereologically-based counts of neurofibrillary tangles, neurons and reactive glia, and morphological analyses of axons were performed in the multimodal association cortex lining the superior temporal sulcus. Levels of synaptic integrity markers, and soluble monomeric and multimeric amyloid-β and tau species were measured. Our results indicate that some individuals can accumulate equivalent loads of amyloid-β plaques and tangles to those found in demented Alzheimer's cases without experiencing dementia. Analyses revealed four main phenotypic differences among these two groups: (i) mismatches had striking preservation of neuron numbers, synaptic markers and axonal geometry compared to demented cases; (ii) demented cases had significantly higher burdens of fibrillar thioflavin-S-positive plaques and of oligomeric amyloid-β deposits reactive to conformer-specific antibody NAB61 than mismatches; (iii) strong and selective accumulation of hyperphosphorylated soluble tau multimers into the synaptic compartment was noted in demented cases compared with controls but not in mismatches; and (iv) the robust glial activation accompanying amyloid-β and tau pathologies in

  15. Differentiation of rat brown adipocytes during late foetal development: role of insulin-like growth factor I.

    PubMed Central

    Teruel, T; Valverde, A M; Alvarez, A; Benito, M; Lorenzo, M

    1995-01-01

    Rat brown adipocytes at day 22 of foetal development showed greater size, higher mitochondria content and larger amounts of lipids, as determined by flow cytometry, than 20-day foetal cells. Simultaneously, an inhibition on the percentage of brown adipocytes into S+G2/M phases of the cell cycle was observed between days 20 and 22 of foetal development. The expression of several adipogenesis-related genes, such as fatty acid synthase, malic enzyme, glucose-6-phosphate dehydrogenase and insulin-regulated glucose transporter, increased at the end of foetal life in brown adipose tissue. In addition, the lipogenic enzyme activities and the lipogenic flux increased during late foetal development, resulting in mature brown adipocytes showing a multilocular fat droplet phenotype. Concurrently, brown adipocytes induced the expression of the uncoupling protein (UP) mRNA and UP protein, as visualized by immunofluorescence. The three isoforms of CCAAT enhancer-binding proteins (C/EBPs) were expressed at the mRNA level in brown adipose tissue at day 20. C/EBP alpha decreased and C/EBP beta and delta increased their expression between days 20 and 22 of foetal development, respectively. Brown adipose tissue constitutively expressed insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNAs. Moreover, IGF-IR mRNA content increased between days 20 and 22 in parallel with the occurrence of tissue differentiation. Images Figure 2 Figure 3 Figure 4 PMID:7575409

  16. Foetal age determination and development in elephants.

    PubMed

    Hildebrandt, Thomas; Drews, Barbara; Gaeth, Ann P; Goeritz, Frank; Hermes, Robert; Schmitt, Dennis; Gray, Charlie; Rich, Peter; Streich, Wolf Juergen; Short, Roger V; Renfree, Marilyn B

    2007-02-01

    Elephants have the longest pregnancy of all mammals, with an average gestation of around 660 days, so their embryonic and foetal development have always been of special interest. Hitherto, it has only been possible to estimate foetal ages from theoretical calculations based on foetal mass. The recent development of sophisticated ultrasound procedures for elephants has now made it possible to monitor the growth and development of foetuses of known gestational age conceived in captivity from natural matings or artificial insemination. We have studied the early stages of pregnancy in 10 captive Asian and 9 African elephants by transrectal ultrasound. Measurements of foetal crown-rump lengths have provided the first accurate growth curves, which differ significantly from the previous theoretical estimates based on the cube root of foetal mass. We have used these to age 22 African elephant foetuses collected during culling operations. Pregnancy can be first recognized ultrasonographically by day 50, the presumptive yolk sac by about day 75 and the zonary placenta by about day 85. The trunk is first recognizable by days 85-90 and is distinct by day 104, while the first heartbeats are evident from around day 80. By combining ultrasonography and morphology, we have been able to produce the first reliable criteria for estimating gestational age and ontological development of Asian and African elephant foetuses during the first third of gestation. PMID:17164195

  17. Distinct phenotypes in zebrafish models of human startle disease☆

    PubMed Central

    Ganser, Lisa R.; Yan, Qing; James, Victoria M.; Kozol, Robert; Topf, Maya; Harvey, Robert J.; Dallman, Julia E.

    2013-01-01

    Startle disease is an inherited neurological disorder that causes affected individuals to suffer noise- or touch-induced non-epileptic seizures, excessive muscle stiffness and neonatal apnea episodes. Mutations known to cause startle disease have been identified in glycine receptor subunit (GLRA1 and GLRB) and glycine transporter (SLC6A5) genes, which serve essential functions at glycinergic synapses. Despite the significant successes in identifying startle disease mutations, many idiopathic cases remain unresolved. Exome sequencing in these individuals will identify new candidate genes. To validate these candidate disease genes, zebrafish is an ideal choice due to rapid knockdown strategies, accessible embryonic stages, and stereotyped behaviors. The only existing zebrafish model of startle disease, bandoneon (beo), harbors point mutations in glrbb (one of two zebrafish orthologs of human GLRB) that cause compromised glycinergic transmission and touch-induced bilateral muscle contractions. In order to further develop zebrafish as a model for startle disease, we sought to identify common phenotypic outcomes of knocking down zebrafish orthologs of two known startle disease genes, GLRA1 and GLRB, using splice site-targeted morpholinos. Although both morphants were expected to result in phenotypes similar to the zebrafish beo mutant, our direct comparison demonstrated that while both glra1 and glrbb morphants exhibited embryonic spasticity, only glrbb morphants exhibited bilateral contractions characteristic of beo mutants. Likewise, zebrafish over-expressing a dominant startle disease mutation (GlyR α1R271Q) exhibited spasticity but not bilateral contractions. Since GlyR βb can interact with GlyR α subunits 2–4 in addition to GlyR α1, loss of the GlyR βb subunit may produce more severe phenotypes by affecting multiple GlyR subtypes. Indeed, immunohistochemistry of glra1 morphants suggests that in zebrafish, alternate GlyR α subunits can compensate for the

  18. Identification of a human recent thymic emigrant phenotype

    PubMed Central

    McFarland, Richard D.; Douek, Daniel C.; Koup, Richard A.; Picker, Louis J.

    2000-01-01

    The ability to measure human thymic output would be an invaluable tool for the study of the development of the naïve T cell repertoire, as well as naïve T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naïve T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that αE integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8+/CD4− thymocytes and also defines a distinct subset of naïve CD8+ T cells in the periphery. The latter subset is differentiated from circulating CD103+ mucosa-associated memory T cells by its naïve T cell phenotype (CD45RO−, CD62Lbright, CD27bright, CD11adim, CD95dim) and its high concentration of TREC. Indeed, sorted CD103+ naïve CD8+ cells display higher levels of TREC than their CD103− naïve counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis. PMID:10737767

  19. Nodal signaling promotes a tumorigenic phenotype in human breast cancer.

    PubMed

    Kirsammer, Gina; Strizzi, Luigi; Margaryan, Naira V; Gilgur, Alina; Hyser, Matthew; Atkinson, Janis; Kirschmann, Dawn A; Seftor, Elisabeth A; Hendrix, Mary J C

    2014-12-01

    The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation. PMID:25073112

  20. Nodal signaling promotes a tumorigenic phenotype in human breast cancer

    PubMed Central

    Kirsammer, Gina; Strizzi, Luigi; Margaryan, Naira V.; Gilgur, Alina; Hyser, Matthew; Atkinson, Janis; Kirschmann, Dawn A.; Seftor, Elisabeth A.; Hendrix, Mary J.C.

    2014-01-01

    The Ras-ERK pathway is deregulated in approximately a third of human cancers, particularly those of epithelial origin. In aggressive, triple-negative, basal-like breast cancers, most tumors display increased MEK and ERK phosphorylation and exhibit a gene expression profile characteristic of Kras or EGFR mutant tumors; however, Ras family genetic mutations are uncommon in triple-negative breast cancer and EGFR mutations account for only a subset of these tumors. Therefore, the upstream events that activate MAPK signaling and promote tumor aggression in triple-negative breast cancers remain poorly defined. We have previously shown that a secreted TGF-β family signaling ligand, Nodal, is expressed in breast cancer in correlation with disease progression. Here we highlight key findings demonstrating that Nodal is required in aggressive human breast cancer cells to activate ERK signaling and downstream tumorigenic phenotypes both in vitro and in vivo. Experimental knockdown of Nodal signaling downregulates ERK activity, resulting in loss of c-myc, upregulation of p27, G1 cell cycle arrest, increased apoptosis and decreased tumorigenicity. The data suggest that ERK activation by Nodal signaling regulates c-myc and p27 proteins post-translationally and that this cascade is essential for aggressive breast tumor behavior in vivo. As the MAPK pathway is an important target for treating triple-negative breast cancers, upstream Nodal signaling may represent a promising target for breast cancer diagnosis and combined therapies aimed at blocking ERK pathway activation. PMID:25073112

  1. Foetal placental blood flow in the lamb

    PubMed Central

    Faber, J. Job; Green, Thomas J.

    1972-01-01

    1. Fifteen sheep foetuses of 1·5-5·2 kg body weight were prepared with indwelling arterial and venous catheters for experimentation one to six days later. 2. Unanaesthetized foetuses were found to have mean arterial and central venous blood pressures of 40 ± 1·5 (S.E. of mean) and 2·0 ± 0·3 (S.E. of mean) mm Hg respectively, compared to intra-uterine pressure. Intra-uterine pressure was 16 ± 0·8 (S.E. of mean) mm Hg with respect to atmospheric pressure at mid-uterine level. 3. Mean placental blood flow of the foetuses was 199 ± 20 (S.E. of mean) ml./(min.kg body wt.). Mean cardiac output in eleven of the foetuses was 658 ± 102 (S.E. of mean) ml./(min.kg). 4. Mean foetal and maternal colloid osmotic pressures were 17·5 ± 0·7 (S.E. of mean) and 20·5 ± 0·6 (S.E. of mean) mm Hg respectively at 38° C. 5. Intravenous infusions into six ewes of 1·8 mole of mannitol and 0·4 mole of NaCl resulted in significant increases in foetal plasma osmolarity, sodium, potassium, and haemoglobin concentrations, without detectable transfer of mannitol to the foetal circulation. 6. In the sheep placenta there is osmotic and hydrostatic equilibration of water. As a consequence, there should be an interaction between foetal placental blood flow and foetal water exchange with the maternal circulation. It was concluded that this interaction tends to stabilize foetal placental blood flow. PMID:5039279

  2. Machine learning for the automatic localisation of foetal body parts in cine-MRI scans

    NASA Astrophysics Data System (ADS)

    Bowles, Christopher; Nowlan, Niamh C.; Hayat, Tayyib T. A.; Malamateniou, Christina; Rutherford, Mary; Hajnal, Joseph V.; Rueckert, Daniel; Kainz, Bernhard

    2015-03-01

    Being able to automate the location of individual foetal body parts has the potential to dramatically reduce the work required to analyse time resolved foetal Magnetic Resonance Imaging (cine-MRI) scans, for example, for use in the automatic evaluation of the foetal development. Currently, manual preprocessing of every scan is required to locate body parts before analysis can be performed, leading to a significant time overhead. With the volume of scans becoming available set to increase as cine-MRI scans become more prevalent in clinical practice, this stage of manual preprocessing is a bottleneck, limiting the data available for further analysis. Any tools which can automate this process will therefore save many hours of research time and increase the rate of new discoveries in what is a key area in understanding early human development. Here we present a series of techniques which can be applied to foetal cine-MRI scans in order to first locate and then differentiate between individual body parts. A novel approach to maternal movement suppression and segmentation using Fourier transforms is put forward as a preprocessing step, allowing for easy extraction of short movements of individual foetal body parts via the clustering of optical flow vector fields. These body part movements are compared to a labelled database and probabilistically classified before being spatially and temporally combined to give a final estimate for the location of each body part.

  3. Phenotypical characterization of regulatory T cells in humans and rodents.

    PubMed

    Rodríguez-Perea, A L; Arcia, E D; Rueda, C M; Velilla, P A

    2016-09-01

    Regulatory T cells (Tregs ) constitute a fascinating subpopulation of CD4(+) T cells due to their ability to limit the immune response against self and non-self antigens. Murine models and antibodies directed against surface and intracellular molecules have allowed elucidation of the mechanisms that govern their development and function. However, these markers used to their classification lack of specificity, as they can be expressed by activated T cells. Similarly, there are slight differences between animal models, in steady state and pathological conditions, anatomical localization and strategy of analysis by flow cytometry. Here, we revised the most common markers utilized for Treg typification by flow cytometry such as CD25, forkhead box protein 3 (FoxP3) and CD127, along with our data obtained in different body compartments of humans, mice and rats. Furthermore, we revised and determined the expression of other molecules important for the phenotypical characterization of Treg cells. We draw attention to the drawbacks of those markers used in chronic states of inflammation. However, until a specific marker for the identification of Tregs is discovered, the best combination of markers will depend upon the tissue or the degree of inflammation from which Tregs derive. PMID:27124481

  4. Generating Phenotypical Erroneous Human Behavior to Evaluate Human-automation Interaction Using Model Checking

    PubMed Central

    Bolton, Matthew L.; Bass, Ellen J.; Siminiceanu, Radu I.

    2012-01-01

    Breakdowns in complex systems often occur as a result of system elements interacting in unanticipated ways. In systems with human operators, human-automation interaction associated with both normative and erroneous human behavior can contribute to such failures. Model-driven design and analysis techniques provide engineers with formal methods tools and techniques capable of evaluating how human behavior can contribute to system failures. This paper presents a novel method for automatically generating task analytic models encompassing both normative and erroneous human behavior from normative task models. The generated erroneous behavior is capable of replicating Hollnagel’s zero-order phenotypes of erroneous action for omissions, jumps, repetitions, and intrusions. Multiple phenotypical acts can occur in sequence, thus allowing for the generation of higher order phenotypes. The task behavior model pattern capable of generating erroneous behavior can be integrated into a formal system model so that system safety properties can be formally verified with a model checker. This allows analysts to prove that a human-automation interactive system (as represented by the model) will or will not satisfy safety properties with both normative and generated erroneous human behavior. We present benchmarks related to the size of the statespace and verification time of models to show how the erroneous human behavior generation process scales. We demonstrate the method with a case study: the operation of a radiation therapy machine. A potential problem resulting from a generated erroneous human action is discovered. A design intervention is presented which prevents this problem from occurring. We discuss how our method could be used to evaluate larger applications and recommend future paths of development. PMID:23105914

  5. Evolutionary history of human disease genes reveals phenotypic connections and comorbidity among genetic diseases

    NASA Astrophysics Data System (ADS)

    Park, Solip; Yang, Jae-Seong; Kim, Jinho; Shin, Young-Eun; Hwang, Jihye; Park, Juyong; Jang, Sung Key; Kim, Sanguk

    2012-10-01

    The extent to which evolutionary changes have impacted the phenotypic relationships among human diseases remains unclear. In this work, we report that phenotypically similar diseases are connected by the evolutionary constraints on human disease genes. Human disease groups can be classified into slowly or rapidly evolving classes, where the diseases in the slowly evolving class are enriched with morphological phenotypes and those in the rapidly evolving class are enriched with physiological phenotypes. Our findings establish a clear evolutionary connection between disease classes and disease phenotypes for the first time. Furthermore, the high comorbidity found between diseases connected by similar evolutionary constraints enables us to improve the predictability of the relative risk of human diseases. We find the evolutionary constraints on disease genes are a new layer of molecular connection in the network-based exploration of human diseases.

  6. Linking Human Diseases to Animal Models Using Ontology-Based Phenotype Annotation

    PubMed Central

    Mungall, Christopher J.; Ashburner, Michael; Westerfield, Monte; Lewis, Suzanna E.

    2009-01-01

    Scientists and clinicians who study genetic alterations and disease have traditionally described phenotypes in natural language. The considerable variation in these free-text descriptions has posed a hindrance to the important task of identifying candidate genes and models for human diseases and indicates the need for a computationally tractable method to mine data resources for mutant phenotypes. In this study, we tested the hypothesis that ontological annotation of disease phenotypes will facilitate the discovery of new genotype-phenotype relationships within and across species. To describe phenotypes using ontologies, we used an Entity-Quality (EQ) methodology, wherein the affected entity (E) and how it is affected (Q) are recorded using terms from a variety of ontologies. Using this EQ method, we annotated the phenotypes of 11 gene-linked human diseases described in Online Mendelian Inheritance in Man (OMIM). These human annotations were loaded into our Ontology-Based Database (OBD) along with other ontology-based phenotype descriptions of mutants from various model organism databases. Phenotypes recorded with this EQ method can be computationally compared based on the hierarchy of terms in the ontologies and the frequency of annotation. We utilized four similarity metrics to compare phenotypes and developed an ontology of homologous and analogous anatomical structures to compare phenotypes between species. Using these tools, we demonstrate that we can identify, through the similarity of the recorded phenotypes, other alleles of the same gene, other members of a signaling pathway, and orthologous genes and pathway members across species. We conclude that EQ-based annotation of phenotypes, in conjunction with a cross-species ontology, and a variety of similarity metrics can identify biologically meaningful similarities between genes by comparing phenotypes alone. This annotation and search method provides a novel and efficient means to identify gene candidates

  7. An artery accompanying the sciatic nerve (arteria comitans nervi ischiadici) and the position of the hip joint: a comparative histological study using chick, mouse, and human foetal specimens.

    PubMed

    Ishizawa, A; Hayashi, S; Nasu, H; Abe, H; Rodríguez-Vázquez, J F; Murakami, G

    2013-02-01

    Birds and reptiles always carry a long and thick artery accompanying the sciatic nerve (i.e., the sciatic artery), whereas mammals do not. We attempted to demonstrate a difference in courses of the nerve and artery in fetuses in relation with the hip joint posture. Eight mid-term human fetuses (15-18 weeks), five mouse fetuses (E18) and five chick embryos (11 days after incubation) were examined histologically. Thin feeding arteries in the sciatic nerve were consistently observed in human fetuses in spite of the long, inferiorly curved course of the nerve around the ischium. The tissue around the human sciatic nerve was not so tight because of the medial and inferior shift of the nerve away from the hip joint. The fetal hip joint position differed among the species, being highly flexed in humans and almost at right angle flexion in mice and chicks. Because of deep adduction of the hip joint in the mouse, the knee was located near the midline of the body. The mouse sciatic nerve ran through the tight tissue along the head of the femur, whereas the chick nerve ran through the loose space even in the gluteal region. In birds, evolution of the pelvis including the hip joint without adduction seemed to make the arterial development possible. In mammals, highly flexed or adducted hip joint seemed to be one of the disturbing factors against development of the long and thick artery. A slight change in posture may cause significant arterial variation. PMID:23749710

  8. Implications of the apportionment of human genetic diversity for the apportionment of human phenotypic diversity

    PubMed Central

    Edge, Michael D.; Rosenberg, Noah A.

    2015-01-01

    Researchers in many fields have considered the meaning of two results about genetic variation for concepts of “race.” First, at most genetic loci, apportionments of human genetic diversity find that worldwide populations are genetically similar. Second, when multiple genetic loci are examined, it is possible to distinguish people with ancestry from different geographical regions. These two results raise an important question about human phenotypic diversity: To what extent do populations typically differ on phenotypes determined by multiple genetic loci? It might be expected that such phenotypes follow the pattern of similarity observed at individual loci. Alternatively, because they have a multilocus genetic architecture, they might follow the pattern of greater differentiation suggested by multilocus ancestry inference. To address the question, we extend a well-known classification model of Edwards (2003) by adding a selectively neutral quantitative trait. Using the extended model, we show, in line with previous work in quantitative genetics, that regardless of how many genetic loci influence the trait, one neutral trait is approximately as informative about ancestry as a single genetic locus. The results support the relevance of single-locus genetic-diversity partitioning for predictions about phenotypic diversity. PMID:25677859

  9. Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry.

    PubMed

    Leipold, Michael D; Newell, Evan W; Maecker, Holden T

    2015-01-01

    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays. PMID:26420710

  10. Multiparameter Phenotyping of Human PBMCs Using Mass Cytometry

    PubMed Central

    Leipold, Michael D.; Newell, Evan W.; Maecker, Holden T.

    2016-01-01

    The standard for single-cell analysis of phenotype and function in recent decades has been fluorescence flow cytometry. Mass cytometry is a newer technology that uses heavy metal ions, rather than fluorochromes, as labels for probes such as antibodies. The binding of these ion-labeled probes to cells is quantitated by mass spectrometry. This greatly increases the number of phenotypic and functional markers that can be probed simultaneously. Here, we review topics that must be considered when adapting existing flow cytometry panels to mass cytometry analysis. We present a protocol and representative panels for surface phenotyping and intracellular cytokine staining (ICS) assays. PMID:26420710

  11. Precision Medicine for Continuing Phenotype Expansion of Human Genetic Diseases

    PubMed Central

    Yu, Hui; Zhang, Victor Wei

    2015-01-01

    Determining the exact genetic causes for a patient and providing definite molecular diagnoses are core elements of precision medicine. Individualized patient care is often limited by our current knowledge of disease etiologies and commonly used phenotypic-based diagnostic approach. The broad and incompletely understood phenotypic spectrum of a disease and various underlying genetic heterogeneity also present extra challenges to our clinical practice. With the rapid adaptation of new sequence technology in clinical setting for diagnostic purpose, phenotypic expansions of disease spectrum are becoming increasingly common. Understanding the underlying molecular mechanisms will help us to integrate genomic information into the workup of individualized patient care and make better clinical decisions. PMID:26137492

  12. Foetal ECG recovery using dynamic neural networks.

    PubMed

    Camps-Valls, Gustavo; Martínez-Sober, Marcelino; Soria-Olivas, Emilio; Magdalena-Benedito, Rafael; Calpe-Maravilla, Javier; Guerrero-Martínez, Juan

    2004-07-01

    Non-invasive electrocardiography has proven to be a very interesting method for obtaining information about the foetus state and thus to assure its well-being during pregnancy. One of the main applications in this field is foetal electrocardiogram (ECG) recovery by means of automatic methods. Evident problems found in the literature are the limited number of available registers, the lack of performance indicators, and the limited use of non-linear adaptive methods. In order to circumvent these problems, we first introduce the generation of synthetic registers and discuss the influence of different kinds of noise to the modelling. Second, a method which is based on numerical (correlation coefficient) and statistical (analysis of variance, ANOVA) measures allows us to select the best recovery model. Finally, finite impulse response (FIR) and gamma neural networks are included in the adaptive noise cancellation (ANC) scheme in order to provide highly non-linear, dynamic capabilities to the recovery model. Neural networks are benchmarked with classical adaptive methods such as the least mean squares (LMS) and the normalized LMS (NLMS) algorithms in simulated and real registers and some conclusions are drawn. For synthetic registers, the most determinant factor in the identification of the models is the foetal-maternal signal-to-noise ratio (SNR). In addition, as the electromyogram contribution becomes more relevant, neural networks clearly outperform the LMS-based algorithm. From the ANOVA test, we found statistical differences between LMS-based models and neural models when complex situations (high foetal-maternal and foetal-noise SNRs) were present. These conclusions were confirmed after doing robustness tests on synthetic registers, visual inspection of the recovered signals and calculation of the recognition rates of foetal R-peaks for real situations. Finally, the best compromise between model complexity and outcomes was provided by the FIR neural network. Both

  13. The human gene map for performance and health-related fitness phenotypes: the 2005 update

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The current review presents the 2005 update of the human gene map for physical performance and health-related fitness phenotypes. It is based on peer-reviewed papers published by the end of 2005. The genes and markers with evidence of association or linkage with a performance or fitness phenotype in...

  14. Ethanol Consumption: How Should We Measure It? Achieving Consilience between Human and Animal Phenotypes

    PubMed Central

    Leeman, Robert F.; Heilig, Markus; Cunningham, Christopher L.; Stephens, David N.; Duka, Taheodora; O’Malley, Stephanie S.

    2010-01-01

    There is only modest overlap in the most common alcohol consumption phenotypes measured in animal studies and those typically studied in humans. To address this issue, we identified a number of alcohol consumption phenotypes of importance to the field that have potential for consilience between human and animal models. These phenotypes can be broken down into three categories: 1) abstinence/the decision to drink or abstain; 2) the actual amount of alcohol consumed and 3) heavy drinking. A number of suggestions for human and animal researchers are made in order to address these phenotypes and enhance consilience. Laboratory studies of the decision to drink or abstain are needed in both human and animal research. In human laboratory studies, heavy or binge drinking that meets cut-offs used in epidemiological and clinical trials should be reported. Greater attention to patterns of drinking over time is needed in both animal and human studies. Individual differences pertaining to all consumption phenotypes should be addressed in animal research. Lastly, improved biomarkers need to be developed in future research for use with both humans and animals. Greater precision in estimating blood alcohol levels in the field together with consistent measurement of breath/blood alcohol levels in human laboratory and animal studies provides one means of achieving greater consilience of alcohol consumption phenotypes. PMID:20148775

  15. Humoral immune responses in foetal sheep.

    PubMed Central

    Fahey, K J; Morris, B

    1978-01-01

    A total of fifty-two foetal sheep between 49 and 126 days gestation were injected with polymeric and monomeric flagellin, dinitrophenylated monomeric flagellin, chicken red blood cells, ovalbumin, ferritin, chicken gamma-globulin and the somatic antigens of Salmonella typhimurium in a variety of combinations. Immune responses were followed in these animals by taking serial blood samples from them through indwelling vascular cannulae and measuring the circulating titres of antibody. Of the antigens tested, ferritin induced immune responses in the youngest foetuses. A short time later in gestation, the majority of foetuses responded to chicken red blood cells, polymeric flagellin, monomeric flagellin and dinitrophenylated monomeric flagellin. Only older foetuses responded regularly to chicken gamma-globulin and ovalbumin. However, antibodies to all these antigens were first detected over the relatively short period of development between 64 and 82 days gestation and this made it difficult to define any precise order in the development of immune responsiveness. Of the antigens tested only the somatic antigens of S. typhimurium failed to induce a primary antibody response during foetal life. The character and magnitude of the antibody responses in foetuses changed throughout in utero development. Both the total amount of antibody produced and the duration of the response increased with foetal age. Foetuses younger than 87 days gestation did not synthesize 2-mercaptoethanol resistant antibodies or IgG1 immunoglobulin to any of the antigens tested, whereas most foetuses older than this regularly did so. PMID:711249

  16. Prediction of gene–phenotype associations in humans, mice, and plants using phenologs

    PubMed Central

    2013-01-01

    Background Phenotypes and diseases may be related to seemingly dissimilar phenotypes in other species by means of the orthology of underlying genes. Such “orthologous phenotypes,” or “phenologs,” are examples of deep homology, and may be used to predict additional candidate disease genes. Results In this work, we develop an unsupervised algorithm for ranking phenolog-based candidate disease genes through the integration of predictions from the k nearest neighbor phenologs, comparing classifiers and weighting functions by cross-validation. We also improve upon the original method by extending the theory to paralogous phenotypes. Our algorithm makes use of additional phenotype data — from chicken, zebrafish, and E. coli, as well as new datasets for C. elegans — establishing that several types of annotations may be treated as phenotypes. We demonstrate the use of our algorithm to predict novel candidate genes for human atrial fibrillation (such as HRH2, ATP4A, ATP4B, and HOPX) and epilepsy (e.g., PAX6 and NKX2-1). We suggest gene candidates for pharmacologically-induced seizures in mouse, solely based on orthologous phenotypes from E. coli. We also explore the prediction of plant gene–phenotype associations, as for the Arabidopsis response to vernalization phenotype. Conclusions We are able to rank gene predictions for a significant portion of the diseases in the Online Mendelian Inheritance in Man database. Additionally, our method suggests candidate genes for mammalian seizures based only on bacterial phenotypes and gene orthology. We demonstrate that phenotype information may come from diverse sources, including drug sensitivities, gene ontology biological processes, and in situ hybridization annotations. Finally, we offer testable candidates for a variety of human diseases, plant traits, and other classes of phenotypes across a wide array of species. PMID:23800157

  17. Phenotypic and functional consequences of herpesvirus saimiri infection of human CD8+ cytotoxic T lymphocytes.

    PubMed Central

    Berend, K R; Jung, J U; Boyle, T J; DiMaio, J M; Mungal, S A; Desrosiers, R C; Lyerly, H K

    1993-01-01

    Herpesvirus saimiri (HVS) was used to infect and transform human CD8+ cytotoxic T lymphocytes (CTL), and the phenotypic and functional consequences of HVS infection of CD8+ T lymphocytes were investigated. HVS-transformed CTL no longer require antigen restimulation yet maintain their phenotype and HLA-restricted cytolytic function and specificity. The ability of HVS to transform CTL may have an important role in the functional analysis of human antigen-specific CTL. Images PMID:8396687

  18. Genotype-phenotype associations and human eye color.

    PubMed

    White, Désirée; Rabago-Smith, Montserrat

    2011-01-01

    Although eye color is usually modeled as a simple, Mendelian trait, further research and observation has indicated that eye color does not follow the classical paths of inheritance. Eye color phenotypes demonstrate both epistasis and incomplete dominance. Although there are about 16 different genes responsible for eye color, it is mostly attributed to two adjacent genes on chromosome 15, hect domain and RCC1-like domain-containing protein 2 (HERC2) and ocular albinism (that is, oculocutaneous albinism II (OCA2)). An intron in HERC2 contains the promoter region for OCA2, affecting its expression. Therefore, single-nucleotide polymorphisms in either of these two genes have a large role in the eye color of an individual. Furthermore, with all genetic expression, aberration also occurs. Some individuals may express two phenotypes--one in each eye--or a complete lack of pigmentation, ocular albinism. In addition, the evolutionary and population roles of the different expressions are significant. PMID:20944644

  19. Analysis of the human diseasome using phenotype similarity between common, genetic, and infectious diseases

    PubMed Central

    Hoehndorf, Robert; Schofield, Paul N.; Gkoutos, Georgios V.

    2015-01-01

    Phenotypes are the observable characteristics of an organism arising from its response to the environment. Phenotypes associated with engineered and natural genetic variation are widely recorded using phenotype ontologies in model organisms, as are signs and symptoms of human Mendelian diseases in databases such as OMIM and Orphanet. Exploiting these resources, several computational methods have been developed for integration and analysis of phenotype data to identify the genetic etiology of diseases or suggest plausible interventions. A similar resource would be highly useful not only for rare and Mendelian diseases, but also for common, complex and infectious diseases. We apply a semantic text-mining approach to identify the phenotypes (signs and symptoms) associated with over 6,000 diseases. We evaluate our text-mined phenotypes by demonstrating that they can correctly identify known disease-associated genes in mice and humans with high accuracy. Using a phenotypic similarity measure, we generate a human disease network in which diseases that have similar signs and symptoms cluster together, and we use this network to identify closely related diseases based on common etiological, anatomical as well as physiological underpinnings. PMID:26051359

  20. Analysis of the human diseasome using phenotype similarity between common, genetic, and infectious diseases

    NASA Astrophysics Data System (ADS)

    Hoehndorf, Robert; Schofield, Paul N.; Gkoutos, Georgios V.

    2015-06-01

    Phenotypes are the observable characteristics of an organism arising from its response to the environment. Phenotypes associated with engineered and natural genetic variation are widely recorded using phenotype ontologies in model organisms, as are signs and symptoms of human Mendelian diseases in databases such as OMIM and Orphanet. Exploiting these resources, several computational methods have been developed for integration and analysis of phenotype data to identify the genetic etiology of diseases or suggest plausible interventions. A similar resource would be highly useful not only for rare and Mendelian diseases, but also for common, complex and infectious diseases. We apply a semantic text-mining approach to identify the phenotypes (signs and symptoms) associated with over 6,000 diseases. We evaluate our text-mined phenotypes by demonstrating that they can correctly identify known disease-associated genes in mice and humans with high accuracy. Using a phenotypic similarity measure, we generate a human disease network in which diseases that have similar signs and symptoms cluster together, and we use this network to identify closely related diseases based on common etiological, anatomical as well as physiological underpinnings.

  1. The Foetal 'Mind' as a Reflection of its Inner Self: Evidence from Colour Doppler Ultrasound of Foetal MCA.

    PubMed

    Kachewar, Sushil Ghanshyam; Gandage, Siddappa Gurubalappa

    2012-01-01

    The unborn healthy foetus is looked upon as a blessing by one and all. A plethora of thoughts arise in the brains of expectant parents. But what goes on in the brain of the yet unborn still remains a mystery. 'Foetal mind' is a reflection of functions of its organs of sense, an instrument of knowledge that may even be reduced to machine to demonstrate the effect of sense organs and brain contact. Testimony to this fact are the various waveform patterns obtained non-invasively from the foetal Middle Cerebral Artery (MCA) by using Colour Doppler Ultrasound. Our study, conducted for evaluating the foetal MCA in a rural obstetric population in Maharashtra, India, explains how the MCA - a major artery supplying foetal brain, can give abundant information about foetal heart and foetal stress. When only the foetal heart is stressed by the presence of arrhythmias or ectopic beats, these changes are manifest in the foetal MCA velocity waveform pattern as seen on Colour Doppler study. When the entire foetus is under stress, as in cases of intra uterine growth retardation (IUGR), changes again manifest in the foetal MCA velocity waveform pattern and are designated as the foetal Brain Sparing Effect. Thus scientific evaluation of foetal MCA waveform can objectively demonstrate that the overtly non-communicating foetal brain indeed remains an internal organ of sense and a vital instrument of knowledge to clarify the various effects of sense organs and brain contact. Although the brain parenchyma or cerebral metabolism has not been studied here, cerebral vessels serve as a window to cerebral metabolism, as auto regulatory function of brain leads to changes in haemodynamics of cerebral vessels. Also, like other vessels, MCA mirrors foetal distress and IUGR; but unlike other vessels, e.g. the umbilical or uterine artery, which show these changes in the form of reduction or even reversal of diastolic flow, MCA shows an increase in diastolic component due to brain sparing effect

  2. The Foetal ‘Mind’ as a Reflection of its Inner Self: Evidence from Colour Doppler Ultrasound of Foetal MCA

    PubMed Central

    Kachewar, Sushil Ghanshyam; Gandage, Siddappa Gurubalappa

    2012-01-01

    The unborn healthy foetus is looked upon as a blessing by one and all. A plethora of thoughts arise in the brains of expectant parents. But what goes on in the brain of the yet unborn still remains a mystery. ‘Foetal mind’ is a reflection of functions of its organs of sense, an instrument of knowledge that may even be reduced to machine to demonstrate the effect of sense organs and brain contact. Testimony to this fact are the various waveform patterns obtained non-invasively from the foetal Middle Cerebral Artery (MCA) by using Colour Doppler Ultrasound. Our study, conducted for evaluating the foetal MCA in a rural obstetric population in Maharashtra, India, explains how the MCA - a major artery supplying foetal brain, can give abundant information about foetal heart and foetal stress. When only the foetal heart is stressed by the presence of arrhythmias or ectopic beats, these changes are manifest in the foetal MCA velocity waveform pattern as seen on Colour Doppler study. When the entire foetus is under stress, as in cases of intra uterine growth retardation (IUGR), changes again manifest in the foetal MCA velocity waveform pattern and are designated as the foetal Brain Sparing Effect. Thus scientific evaluation of foetal MCA waveform can objectively demonstrate that the overtly non-communicating foetal brain indeed remains an internal organ of sense and a vital instrument of knowledge to clarify the various effects of sense organs and brain contact. Although the brain parenchyma or cerebral metabolism has not been studied here, cerebral vessels serve as a window to cerebral metabolism, as auto regulatory function of brain leads to changes in haemodynamics of cerebral vessels. Also, like other vessels, MCA mirrors foetal distress and IUGR; but unlike other vessels, e.g. the umbilical or uterine artery, which show these changes in the form of reduction or even reversal of diastolic flow, MCA shows an increase in diastolic component due to brain sparing effect

  3. Phenolyzer: phenotype-based prioritization of candidate genes for human diseases.

    PubMed

    Yang, Hui; Robinson, Peter N; Wang, Kai

    2015-09-01

    Prior biological knowledge and phenotype information may help to identify disease genes from human whole-genome and whole-exome sequencing studies. We developed Phenolyzer (http://phenolyzer.usc.edu), a tool that uses prior information to implicate genes involved in diseases. Phenolyzer exhibits superior performance over competing methods for prioritizing Mendelian and complex disease genes, based on disease or phenotype terms entered as free text. PMID:26192085

  4. Tumor-Associated Neutrophils Show Phenotypic and Functional Divergence in Human Lung Cancer.

    PubMed

    Saha, Shilpi; Biswas, Subhra K

    2016-07-11

    Studies in murine cancer models have demonstrated the phenotypic and functional divergence of neutrophils; however, their role in pro- or anti-tumor responses in human remains elusive. In this issue of Cancer Cell, Singhal et al. report the existence of specialized subsets of neutrophils in human lung cancer with diverging functions. PMID:27411583

  5. Influence of age, irradiation and humanization on NSG mouse phenotypes

    PubMed Central

    Knibbe-Hollinger, Jaclyn S.; Fields, Natasha R.; Chaudoin, Tammy R; Epstein, Adrian A.; Makarov, Edward; Akhter, Sidra P.; Gorantla, Santhi; Bonasera, Stephen J.; Gendelman, Howard E.; Poluektova, Larisa Y.

    2015-01-01

    ABSTRACT Humanized mice are frequently utilized in bench to bedside therapeutic tests to combat human infectious, cancerous and degenerative diseases. For the fields of hematology-oncology, regenerative medicine, and infectious diseases, the immune deficient mice have been used commonly in basic research efforts. Obstacles in true translational efforts abound, as the relationship between mouse and human cells in disease pathogenesis and therapeutic studies requires lengthy investigations. The interplay between human immunity and mouse biology proves ever more complicated when aging, irradiation, and human immune reconstitution are considered. All can affect a range of biochemical and behavioral functions. To such ends, we show age- and irradiation-dependent influences for the development of macrocytic hyper chromic anemia, myelodysplasia, blood protein reductions and body composition changes. Humanization contributes to hematologic abnormalities. Home cage behavior revealed day and dark cycle locomotion also influenced by human cell reconstitutions. Significant age-related day-to-day variability in movement, feeding and drinking behaviors were observed. We posit that this data serves to enable researchers to better design translational studies in this rapidly emerging field of mouse humanization. PMID:26353862

  6. Potassium Channels and Human Epileptic Phenotypes: An Updated Overview

    PubMed Central

    Villa, Chiara; Combi, Romina

    2016-01-01

    Potassium (K+) channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K+ channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K+ channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K+ channels dysfunctions linked to inherited epilepsy in humans and non-human model animals. This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K+ channels in monogenic forms. PMID:27064559

  7. Potassium Channels and Human Epileptic Phenotypes: An Updated Overview.

    PubMed

    Villa, Chiara; Combi, Romina

    2016-01-01

    Potassium (K(+)) channels are expressed in almost every cells and are ubiquitous in neuronal and glial cell membranes. These channels have been implicated in different disorders, in particular in epilepsy. K(+) channel diversity depends on the presence in the human genome of a large number of genes either encoding pore-forming or accessory subunits. More than 80 genes encoding the K(+) channels were cloned and they represent the largest group of ion channels regulating the electrical activity of cells in different tissues, including the brain. It is therefore not surprising that mutations in these genes lead to K(+) channels dysfunctions linked to inherited epilepsy in humans and non-human model animals. This article reviews genetic and molecular progresses in exploring the pathogenesis of different human epilepsies, with special emphasis on the role of K(+) channels in monogenic forms. PMID:27064559

  8. Phenotypic and genotypic characterization of human and nonhuman Escherichia coli.

    PubMed

    Parveen, S; Hodge, N C; Stall, R E; Farrah, S R; Tamplin, M L

    2001-02-01

    Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates. PMID:11228989

  9. Computer analysis of foetal monitoring signals.

    PubMed

    Nunes, Inês; Ayres-de-Campos, Diogo

    2016-01-01

    Five systems for computer analysis of foetal monitoring signals are currently available, incorporating the evaluation of cardiotocographic (CTG) or combined CTG with electrocardiographic ST data. All systems have been integrated with central monitoring stations, allowing the simultaneous monitoring of several tracings on the same computer screen in multiple hospital locations. Computer analysis elicits real-time visual and sound alerts for health care professionals when abnormal patterns are detected, with the aim of prompting a re-evaluation and subsequent clinical action, if considered necessary. Comparison between the CTG analyses provided by the computer and clinical experts has been carried out in all systems, and in three of them, the accuracy of computer alerts in predicting newborn outcomes was evaluated. Comparisons between these studies are hampered by the differences in selection criteria and outcomes. Two of these systems have just completed multicentre randomised clinical trials comparing them with conventional CTG monitoring, and their results are awaited shortly. For the time being, there is limited evidence regarding the impact of computer analysis of foetal monitoring signals on perinatal indicators and on health care professionals' behaviour. PMID:26211832

  10. PHENOstruct: Prediction of human phenotype ontology terms using heterogeneous data sources

    PubMed Central

    Kahanda, Indika; Funk, Christopher; Verspoor, Karin; Ben-Hur, Asa

    2015-01-01

    The human phenotype ontology (HPO) was recently developed as a standardized vocabulary for describing the phenotype abnormalities associated with human diseases. At present, only a small fraction of human protein coding genes have HPO annotations. But, researchers believe that a large portion of currently unannotated genes are related to disease phenotypes. Therefore, it is important to predict gene-HPO term associations using accurate computational methods. In this work we demonstrate the performance advantage of the structured SVM approach which was shown to be highly effective for Gene Ontology term prediction in comparison to several baseline methods. Furthermore, we highlight a collection of informative data sources suitable for the problem of predicting gene-HPO associations, including large scale literature mining data. PMID:26834980

  11. Genetic modeling of ovarian phenotypes in mice for the study of human polycystic ovary syndrome.

    PubMed

    Feng, Yi; Li, Xin; Shao, Ruijin

    2013-01-01

    Polycystic ovary syndrome (PCOS) presents with a range of clinical complications including hyperandrogenism, polycystic ovaries, chronic oligo/anovulation, infertility, and metabolic alterations related to insulin resistance. Because the mechanism by which this disorder develops is poorly understood, information from experimental models of human disease phenotypes may help to define the mechanisms for the initiation and development of PCOS-related pathological events. The establishment of animal models compatible with human PCOS is challenging, and applying the lessons learned from these models to human PCOS is often complicated. In this mini-review we provide examples of currently available genetic mouse models, their ovarian phenotypes, and their possible relationship to different aspects of human PCOS. Because of the practical and ethical limitations of studying PCOS-related events in humans, our understanding of the mechanisms that contribute to the etiology of human PCOS may be enhanced through further study of these transgenic and knockout mouse models. PMID:23390562

  12. Computational evaluation of exome sequence data using human and model organism phenotypes improves diagnostic efficiency

    PubMed Central

    Bone, William P.; Washington, Nicole L.; Buske, Orion J.; Adams, David R.; Davis, Joie; Draper, David; Flynn, Elise D.; Girdea, Marta; Godfrey, Rena; Golas, Gretchen; Groden, Catherine; Jacobsen, Julius; Köhler, Sebastian; Lee, Elizabeth M. J.; Links, Amanda E.; Markello, Thomas C.; Mungall, Christopher J.; Nehrebecky, Michele; Robinson, Peter N.; Sincan, Murat; Soldatos, Ariane G.; Tifft, Cynthia J.; Toro, Camilo; Trang, Heather; Valkanas, Elise; Vasilevsky, Nicole; Wahl, Colleen; Wolfe, Lynne A.; Boerkoel, Cornelius F.; Brudno, Michael; Haendel, Melissa A.; Gahl, William A.; Smedley, Damian

    2016-01-01

    Purpose: Medical diagnosis and molecular or biochemical confirmation typically rely on the knowledge of the clinician. Although this is very difficult in extremely rare diseases, we hypothesized that the recording of patient phenotypes in Human Phenotype Ontology (HPO) terms and computationally ranking putative disease-associated sequence variants improves diagnosis, particularly for patients with atypical clinical profiles. Genet Med 18 6, 608–617. Methods: Using simulated exomes and the National Institutes of Health Undiagnosed Diseases Program (UDP) patient cohort and associated exome sequence, we tested our hypothesis using Exomiser. Exomiser ranks candidate variants based on patient phenotype similarity to (i) known disease–gene phenotypes, (ii) model organism phenotypes of candidate orthologs, and (iii) phenotypes of protein–protein association neighbors. Genet Med 18 6, 608–617. Results: Benchmarking showed Exomiser ranked the causal variant as the top hit in 97% of known disease–gene associations and ranked the correct seeded variant in up to 87% when detectable disease–gene associations were unavailable. Using UDP data, Exomiser ranked the causative variant(s) within the top 10 variants for 11 previously diagnosed variants and achieved a diagnosis for 4 of 23 cases undiagnosed by clinical evaluation. Genet Med 18 6, 608–617. Conclusion: Structured phenotyping of patients and computational analysis are effective adjuncts for diagnosing patients with genetic disorders. Genet Med 18 6, 608–617. PMID:26562225

  13. Comparative Analyses of QTLs Influencing Obesity and Metabolic Phenotypes in Pigs and Humans

    PubMed Central

    Jacobsen, Mette J.; Cirera, Susanna; Kogelman, Lisette J. A.; Bruun, Camilla S.; Mark, Thomas; Jørgensen, Claus B.; Grarup, Niels; Appel, Emil V. R.; Galjatovic, Ehm A. A.; Hansen, Torben; Pedersen, Oluf; Guerin, Maryse; Huby, Thierry; Lesnik, Philipppe; Meuwissen, Theo H. E.; Kadarmideen, Haja N.; Fredholm, Merete

    2015-01-01

    The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel insight that

  14. Key concepts in human genetics: understanding the complex phenotype.

    PubMed

    Gibson, William T

    2009-01-01

    The recent sequencing of a reference human genome has generated a large number of DNA-based tools, which are being used to locate genes that contribute to disease. These tools have also enabled studies of the genetics of non-disease traits such as athletic fitness. Sport scientists should keep in mind three major factors when designing such studies and interpreting the literature. First of all, the methods used to assign a biological trait (be it performance related or disease related) to a specific gene are not as powerful as is commonly believed. Second, the methods used are thought to be more robust for disease-related traits than for normal physical characteristics, likely because there are many more biological factors contributing to the latter. Third, additional levels of variability continue to be uncovered in the human genome; these may ultimately contribute more to physical differences between human beings than the levels studied over the past decade. This introductory chapter will aim to equip the reader with the necessary vocabulary to understand and interpret genetic studies targeted to sport fitness and sport-related injury. PMID:19696504

  15. Regulatory networks define phenotypic classes of human stem cell lines

    PubMed Central

    Müller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

    2008-01-01

    Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks. PMID:18724358

  16. From genotype to human β cell phenotype and beyond

    PubMed Central

    Marchetti, Piero; Syed, Farooq; Suleiman, Mara; Bugliani, Marco; Marselli, Lorella

    2012-01-01

    Polygenic type 2 diabetes mellitus (T2DM) is a multi-factorial disease due to the interplay between genes and the environment. Over the years, several genes/loci have been associated with this type of diabetes, with the majority of them being related to β cell dysfunction. In this review, the available information on how polymorphisms in T2DM-associated genes/loci do directly affect the properties of human islet cells are presented and discussed, including some clinical implications and the role of epigenetic mechanisms. PMID:23073174

  17. Mutational spectrometry without phenotypic selection: human mitochondrial DNA.

    PubMed Central

    Khrapko, K; Coller, H; André, P; Li, X C; Foret, F; Belenky, A; Karger, B L; Thilly, W G

    1997-01-01

    By first separating mutant from nonmutant DNA sequences on the basis of their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that allows observation of point mutations in biological samples at fractions at or above 10-6. Using this method, we have observed the hotspot point mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues. To date, 19 mutants have been isolated, their fractions ranging from 4x10-4 down to the limit of detection. We performed specific tests to determine if the observed signals were artefacts arising from contamination, polymerase errors during PCR or DNA adducts created during the procedure. We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, were included with true mutants in our separation steps and converted to mutants during PCR. We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at least in part from mismatch intermediates or cellular DNA adducts. This technology is therefore sufficient for the observation of the spectrum of point mutations in human mitochondrial DNA and is a tool for discovering the primary causes of these mutations. PMID:9016616

  18. Phenotype expression of human bone cells cultured on implant substrates.

    PubMed

    Locci, P; Becchetti, E; Pugliese, M; Rossi, L; Belcastro, S; Calvitti, M; Pietrarelli, G; Staffolani, N

    1997-09-01

    Bone cells derived from the human jaw were cultured on titanium, titanium coated with hydroxyapatite (THA) or with plasma spray (TPS) to study the behaviour of the cells anchored to implant substrates. Bone cells were cultured in MEM with the addition of [3H]-thymidine to evaluate cellular proliferation, and [3H]-glucosamine to evaluate GAG synthesis and accumulation in the extra-cellular matrix (ECM). Moreover, to study the degradation of GAG bone cells were cultured in the presence of NH4Cl, an amine known to inhibit lysosomal activity. Our results show that TPS is the substrate that favours both cellular proliferation and the accumulation of GAG in the ECM. PMID:9377794

  19. Null mutations in human and mouse orthologs frequently result in different phenotypes

    PubMed Central

    Liao, Ben-Yang; Zhang, Jianzhi

    2008-01-01

    One-to-one orthologous genes of relatively closely related species are widely assumed to have similar functions and cause similar phenotypes when deleted from the genome. Although this assumption is the foundation of comparative genomics and the basis for the use of model organisms to study human biology and disease, its validity is known only from anecdotes rather than from systematic examination. Comparing documented phenotypes of null mutations in humans and mice, we find that >20% of human essential genes have nonessential mouse orthologs. These changes of gene essentiality appear to be associated with adaptive evolution at the protein-sequence, but not gene-expression, level. Proteins localized to the vacuole, a cellular compartment for waste management, are highly enriched among essentiality-changing genes. It is probable that the evolution of the prolonged life history in humans required enhanced waste management for proper cellular function until the time of reproduction, which rendered these vacuole proteins essential and generated selective pressures for their improvement. If our gene sample represents the entire genome, our results would mean frequent changes of phenotypic effects of one-to-one orthologous genes even between relatively closely related species, a possibility that should be considered in comparative genomic studies and in making cross-species inferences of gene function and phenotypic effect. PMID:18458337

  20. Genetic and phenotypic evidence of the Salmonella enterica serotype Enteritidis human-animal interface in Chile.

    PubMed

    Retamal, Patricio; Fresno, Marcela; Dougnac, Catherine; Gutierrez, Sindy; Gornall, Vanessa; Vidal, Roberto; Vernal, Rolando; Pujol, Myriam; Barreto, Marlen; González-Acuña, Daniel; Abalos, Pedro

    2015-01-01

    Salmonella enterica serotype Enteritidis is a worldwide zoonotic agent that has been recognized as a very important food-borne bacterial pathogen, mainly associated with consumption of poultry products. The aim of this work was to determine genotypic and phenotypic evidence of S. Enteritidis transmission among seabirds, poultry and humans in Chile. Genotyping was performed using PCR-based virulotyping, pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Pathogenicity-associated phenotypes were determined with survival to free radicals, acidic pH, starvation, antimicrobial resistance, and survival within human dendritic cells. As result of PCR and PFGE assays, some isolates from the three hosts showed identical genotypic patterns, and through MLST it was determined that all of them belong to sequence type 11. Phenotypic assays show diversity of bacterial responses among isolates. When results were analyzed according to bacterial host, statistical differences were identified in starvation and dendritic cells survival assays. In addition, isolates from seabirds showed the highest rates of resistance to gentamycin, tetracycline, and ampicillin. Overall, the very close genetic and phenotypic traits shown by isolates from humans, poultry, and seabirds suggest the inter-species transmission of S. Enteritidis bacteria between hosts, likely through anthropogenic environmental contamination that determines infection of seabirds with bacteria that are potentially pathogenic for other susceptible organism, including humans. PMID:26029196

  1. Genetic and phenotypic evidence of the Salmonella enterica serotype Enteritidis human-animal interface in Chile

    PubMed Central

    Retamal, Patricio; Fresno, Marcela; Dougnac, Catherine; Gutierrez, Sindy; Gornall, Vanessa; Vidal, Roberto; Vernal, Rolando; Pujol, Myriam; Barreto, Marlen; González-Acuña, Daniel; Abalos, Pedro

    2015-01-01

    Salmonella enterica serotype Enteritidis is a worldwide zoonotic agent that has been recognized as a very important food-borne bacterial pathogen, mainly associated with consumption of poultry products. The aim of this work was to determine genotypic and phenotypic evidence of S. Enteritidis transmission among seabirds, poultry and humans in Chile. Genotyping was performed using PCR-based virulotyping, pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Pathogenicity-associated phenotypes were determined with survival to free radicals, acidic pH, starvation, antimicrobial resistance, and survival within human dendritic cells. As result of PCR and PFGE assays, some isolates from the three hosts showed identical genotypic patterns, and through MLST it was determined that all of them belong to sequence type 11. Phenotypic assays show diversity of bacterial responses among isolates. When results were analyzed according to bacterial host, statistical differences were identified in starvation and dendritic cells survival assays. In addition, isolates from seabirds showed the highest rates of resistance to gentamycin, tetracycline, and ampicillin. Overall, the very close genetic and phenotypic traits shown by isolates from humans, poultry, and seabirds suggest the inter-species transmission of S. Enteritidis bacteria between hosts, likely through anthropogenic environmental contamination that determines infection of seabirds with bacteria that are potentially pathogenic for other susceptible organism, including humans. PMID:26029196

  2. An enzyme linked immunosorbent assay (ELISA) for the determination of the human haptoglobin phenotype

    PubMed Central

    Levy, Nina S.; Vardi, Moshe; Blum, Shany; Miller-Lotan, Rachel; Afinbinder, Yefim; Cleary, Patricia A.; Paterson, Andrew D.; Bharaj, Bhupinder; Snell-Bergeon, Janet K.; Rewers, Marian J.; Lache, Orit; Levy, Andrew P.

    2013-01-01

    Background Haptoglobin (Hp) is an abundant serum protein which binds extracorpuscular hemoglobin (Hb). Two alleles exist in humans for the Hp gene, denoted 1 and 2. Diabetic individuals with the Hp 2-2 genotype are at increased risk of developing vascular complications including heart attack, stroke, and kidney disease. Recent evidence shows that treatment with vitamin E can reduce the risk of diabetic vascular complications by as much as 50% in Hp 2-2 individuals. We sought to develop a rapid and accurate test for Hp phenotype (which is 100% concordant with the three major Hp genotypes) to facilitate widespread diagnostic testing as well as prospective clinical trials. Methods A monoclonal antibody raised against human Hp was shown to distinguish between the three Hp phenotypes in an enzyme linked immunosorbent assay (ELISA). Hp phenotypes obtained in over 8000 patient samples using this ELISA method were compared with those obtained by polyacrylamide gel electrophoresis or the TaqMan PCR method. Results Our analysis showed that the sensitivity and specificity of the ELISA test for Hp 2-2 phenotype is 99.0% and 98.1%, respectively. The positive predictive value and the negative predictive value for Hp 2-2 phenotype is 97.5% and 99.3%, respectively. Similar results were obtained for Hp 2-1 and Hp 1-1 phenotypes. In addition, the ELISA was determined to be more sensitive and specific than the TaqMan method. Conclusions The Hp ELISA represents a user-friendly, rapid and highly accurate diagnostic tool for determining Hp phenotypes. This test will greatly facilitate the typing of thousands of samples in ongoing clinical studies. PMID:23492570

  3. Phenotypic Characterization Analysis of Human Hepatocarcinoma by Urine Metabolomics Approach

    PubMed Central

    Liang, Qun; Liu, Han; Wang, Cong; Li, Binbing

    2016-01-01

    Hepatocarcinoma (HCC) is one of the deadliest cancers in the world and represents a significant disease burden. Better biomarkers are needed for early detection of HCC. Metabolomics was applied to urine samples obtained from HCC patients to discover noninvasive and reliable biomarkers for rapid diagnosis of HCC. Metabolic profiling was performed by LC-Q-TOF-MS in conjunction with multivariate data analysis, machine learning approaches, ingenuity pathway analysis and receiver-operating characteristic curves were used to select the metabolites which were used for the noninvasive diagnosis of HCC. Fifteen differential metabolites contributing to the complete separation of HCC patients from matched healthy controls were identified involving several key metabolic pathways. More importantly, five marker metabolites were effective for the diagnosis of human HCC, achieved a sensitivity of 96.5% and specificity of 83% respectively, could significantly increase the diagnostic performance of the metabolic biomarkers. Overall, these results illustrate the power of the metabolomics technology which has the potential as a non-invasive strategies and promising screening tool to evaluate the potential of the metabolites in the early diagnosis of HCC patients at high risk and provides new insight into pathophysiologic mechanisms. PMID:26805550

  4. Effect of hydroxyapatite-based biomaterials on human osteoblast phenotype.

    PubMed

    Trombelli, L; Penolazzi, L; Torreggiani, E; Farina, R; Lambertini, E; Vecchiatini, R; Piva, R

    2010-03-01

    The present study evaluated human primary osteoblasts and two different osteoblast-like cell lines behaviour when cultured in presence of different hydroxyapatite-based (HA) biomaterials (SINTlife-FIN-CERAMICA S.p.a., Faenza, Italy; Bio-Oss, Geistlich Biomaterials, Woulhusen, Switzerland; Biostite-GABA Vebas, San Giuliano Milanese, MI, Italy), focusing attention on the effect of HA/Biostite in terms of modulation of osteoblastic differentiation. Analysis were about adhesion, proliferation and mineralization activity. Runt-related transcription factor 2 (Runx2), Estrogen Receptor alpha (ERalfa) expression and alkaline phosphatase activity (ALP) were measured as osteoblastic differentiation markers. Determination of viable cells was done with MTT colorimetric assay. Scanning electron microscopy (SEM) analysis was performed on biomaterial-treated cells. All hydroxyapatite-based biomaterials didn't affect cells morphology and viability, whereas only presence of HA/Biostite improved cells adhesion, growth and differentiation. Adhesion and spreading of the primary cells on HA/Biostite were the same showed by two different osteoblast-like cell lines. These results have important implications for both tissue-engineered bone grafts and enhancement of HA implants performance, to develop new teeth's supporting structure therapies and replacement. PMID:20357737

  5. Human regulatory B cells combine phenotypic and genetic hallmarks with a distinct differentiation fate.

    PubMed

    Lin, Wenyu; Cerny, Daniela; Chua, Edmond; Duan, Kaibo; Yi, June Tai Jing; Shadan, Nurhidaya Binte; Lum, Josephine; Maho-Vaillant, Maud; Zolezzi, Francesca; Wong, Siew Cheng; Larbi, Anis; Fink, Katja; Musette, Philippe; Poidinger, Michael; Calbo, Sébastien

    2014-09-01

    Regulatory B cells (B-reg) produce IL-10 and suppress inflammation in both mice and humans, but limited data on the phenotype and function of these cells have precluded detailed assessment of their contribution to host immunity. In this article, we report that human B-reg cannot be defined based on a phenotype composed of conventional B cell markers, and that IL-10 production can be elicited in both the CD27(+) memory population and naive B cell subset after only a brief stimulation in vitro. We therefore sought to obtain a better definition of IL-10-producing human B-regs using a multiparameter analysis of B cell phenotype, function, and gene expression profile. Exposure to CpG and anti-Ig are the most potent stimuli for IL-10 secretion in human B cells, but microarray analysis revealed that human B cells cotreated with these reagents resulted in only ∼0.7% of genes being differentially expressed between IL-10(+) and IL-10(-) cells. Instead, connectivity map analysis revealed that IL-10-secreting B cells are those undergoing specific differentiation toward a germinal center fate, and we identified a CD11c(+) B cell subset that was not capable of producing IL-10 even under optimal conditions. Our findings will assist in the identification of a broader range of human pro-B-reg populations that may represent novel targets for immunotherapy. PMID:25080484

  6. The effect of transposable elements on phenotypic variation: insights from plants to humans.

    PubMed

    Wei, Liya; Cao, Xiaofeng

    2016-01-01

    Transposable elements (TEs), originally discovered in maize as controlling elements, are the main components of most eukaryotic genomes. TEs have been regarded as deleterious genomic parasites due to their ability to undergo massive amplification. However, TEs can regulate gene expression and alter phenotypes. Also, emerging findings demonstrate that TEs can establish and rewire gene regulatory networks by genetic and epigenetic mechanisms. In this review, we summarize the key roles of TEs in fine-tuning the regulation of gene expression leading to phenotypic plasticity in plants and humans, and the implications for adaption and natural selection. PMID:26753674

  7. Frequency of Foetal Anomalies in a Tertiary Care Centre

    PubMed Central

    Babu, Rameswarapu Suman; Pasula, Sujatha

    2013-01-01

    Objective: The present study was undertaken to explore the incidence of congenital foetal anomalies and the advantages of ultrasonography in detecting the foetal anomalies during the antenatal period. Method: We focused our study on 1000 consecutive pregnancies that came for check up in the second and third trimesters, with major or minor clinically relevant malformations which were detectable by ultrasonography. Results: The analysis revealed that they were 38 foetal anomalies in 37 foetuses. One had multiple anomalies, with the highest incidence of neural tube defects. There was also significant correlation with consanguinity. Conclusion: The overall incidence of congenital foetal anomalies in the present study was 3.8%.This might be probably due to environmental pollution, radiation, exposure to different chemicals and teratogenic drugs. PMID:23998044

  8. The multiscale backbone of the human phenotype network based on biological pathways

    PubMed Central

    2014-01-01

    Background Networks are commonly used to represent and analyze large and complex systems of interacting elements. In systems biology, human disease networks show interactions between disorders sharing common genetic background. We built pathway-based human phenotype network (PHPN) of over 800 physical attributes, diseases, and behavioral traits; based on about 2,300 genes and 1,200 biological pathways. Using GWAS phenotype-to-genes associations, and pathway data from Reactome, we connect human traits based on the common patterns of human biological pathways, detecting more pleiotropic effects, and expanding previous studies from a gene-centric approach to that of shared cell-processes. Results The resulting network has a heavily right-skewed degree distribution, placing it in the scale-free region of the network topologies spectrum. We extract the multi-scale information backbone of the PHPN based on the local densities of the network and discarding weak connection. Using a standard community detection algorithm, we construct phenotype modules of similar traits without applying expert biological knowledge. These modules can be assimilated to the disease classes. However, we are able to classify phenotypes according to shared biology, and not arbitrary disease classes. We present examples of expected clinical connections identified by PHPN as proof of principle. Conclusions We unveil a previously uncharacterized connection between phenotype modules and discuss potential mechanistic connections that are obvious only in retrospect. The PHPN shows tremendous potential to become a useful tool both in the unveiling of the diseases’ common biology, and in the elaboration of diagnosis and treatments. PMID:24460644

  9. Regulatory mechanism of human vascular smooth muscle cell phenotypic transformation induced by NELIN.

    PubMed

    Pei, Changan; Qin, Shiyong; Wang, Minghai; Zhang, Shuguang

    2015-11-01

    Vascular disorders, including hypertension, atherosclerosis and restenosis, arise from dysregulation of vascular smooth muscle cell (VSMC) differentiation, which can be controlled by regulatory factors. The present study investigated the regulatory mechanism of the phenotypic transformation of human VSMCs by NELIN in order to evaluate its potential as a preventive and therapeutic of vascular disorders. An in vitro model of NELIN‑overexpressing VSMCs was prepared by transfection with a lentiviral (LV) vector (NELIN‑VSMCs) and NELIN was slienced using an a lentiviral vector with small interfering (si)RNA in another group (LV‑NELIN‑siRNA‑VSMCs). The effects of NELIN overexpression or knockdown on the phenotypic transformation of human VSMCs were observed, and its regulatory mechanism was studied. Compared with the control group, cells in the NELIN‑VSMCs group presented a contractile phenotype with a significant increase of NELIN mRNA, NELIN protein, smooth muscle (SM)α‑actin and total Ras homolog gene family member A (RhoA) protein expression. The intra‑nuclear translocation of SMα‑actin‑serum response factor (SMα‑actin‑SRF) occurred in these cells simultaneously. Following exposure to Rho kinsase inhibitor Y‑27632, SRF and SMα‑actin expression decreased. However, cells in the LV‑NELIN‑siRNA‑VSMCs group presented a synthetic phenotype, and the expression of NELIN mRNA, NELIN protein, SMα‑actin protein and total RhoA protein was decreased. The occurrence of SRF extra‑nuclear translocation was observed. In conclusion, the present study suggested that NELIN was able to activate regulatory factors of SMα‑actin, RhoA and SRF successively in human VSMCs cultured in vitro. Furthermore, NELIN‑induced phenotypic transformation of human VSMCs was regulated via the RhoA/SRF signaling pathway. The results of the present study provide a foundation for the use of NELIN in preventive and therapeutic treatment of vascular remodeling

  10. Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.

    PubMed

    Tarique, Abdullah A; Logan, Jayden; Thomas, Emma; Holt, Patrick G; Sly, Peter D; Fantino, Emmanuelle

    2015-11-01

    Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1β, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli. PMID:25870903

  11. The role of DNA insertions in phenotypic differentiation between humans and other primates.

    PubMed

    Hellen, Elizabeth H B; Kern, Andrew D

    2015-04-01

    What makes us human is one of the most interesting and enduring questions in evolutionary biology. To assist in answering this question, we have identified insertions in the human genome which cannot be found in five comparison primate species: Chimpanzee, gorilla, orangutan, gibbon, and macaque. A total of 21,269 nonpolymorphic human-specific insertions were identified, of which only 372 were found in exons. Any function conferred by the remaining 20,897 is likely to be regulatory. Many of these insertions are likely to have been fitness neutral; however, a small number has been identified in genes showing signs of positive selection. Insertions found within positively selected genes show associations to neural phenotypes, which were also enriched in the whole data set. Other phenotypes that are found to be enriched in the data set include dental and sensory perception-related phenotypes, features which are known to differ between humans and other apes. The analysis provides several likely candidates, either genes or regulatory regions, which may be involved in the processes that differentiate humans from other apes. PMID:25635043

  12. Phenotypes of Myopathy-Related Beta-Tropomyosin Mutants in Human and Mouse Tissue Cultures

    PubMed Central

    Abdul-Hussein, Saba; Rahl, Karin; Moslemi, Ali-Reza; Tajsharghi, Homa

    2013-01-01

    Mutations in TPM2 result in a variety of myopathies characterised by variable clinical and morphological features. We used human and mouse cultured cells to study the effects of β-TM mutants. The mutants induced a range of phenotypes in human myoblasts, which generally changed upon differentiation to myotubes. Human myotubes transfected with the E41K-β-TMEGFP mutant showed perinuclear aggregates. The G53ins-β-TMEGFP mutant tended to accumulate in myoblasts but was incorporated into filamentous structures of myotubes. The K49del-β-TMEGFP and E122K-β-TMEGFP mutants induced the formation of rod-like structures in human cells. The N202K-β-TMEGFP mutant failed to integrate into thin filaments and formed accumulations in myotubes. The accumulation of mutant β-TMEGFP in the perinuclear and peripheral areas of the cells was the striking feature in C2C12. We demonstrated that human tissue culture is a suitable system for studying the early stages of altered myofibrilogenesis and morphological changes linked to myopathy-related β-TM mutants. In addition, the histopathological phenotype associated with expression of the various mutant proteins depends on the cell type and varies with the maturation of the muscle cell. Further, the phenotype is a combinatorial effect of the specific amino acid change and the temporal expression of the mutant protein. PMID:24039757

  13. Tumourigenic phenotypes of human melanoma cell lines in nude mice determined by an active antitumour mechanism.

    PubMed Central

    Jacubovich, R.; Cabrillat, H.; Gerlier, D.; Bailly, M.; Doré, J. F.

    1985-01-01

    Ten human melanoma cell lines (HMCL) were tested for their ability to grow subcutaneously in nude mice. Using a standard inoculum, the HMCL could be characterized by their highly, fairly or poorly xenografting phenotype. These phenotypes were stable and the phenotype of one HMCL was recovered within cell clones derived from it. The role of nude mice natural defences in the expression of HMCL xenografting phenotypes was studied. Sublethal whole body irradiation and silica pretreatment of recipients enabled poorly tumourigenic HMCL to grow in most animals without affecting their splenic NK activity. Admixture of BCG or MDP encapsulated in liposomes with highly tumourigenic HMCL resulted in the abrogation of tumour growth in naive nude mice. The long lasting abrogating of NK activity in vivo by treatment with anti-asialo-GM1 anti-serum did not enhance the growth of a poorly tumourigenic HMCL. The HMCL were found to be resistant to in vitro murine NK activity. These results showed that the expression of the HMCL xenografting phenotypes could be controlled by the nude mice natural defences. NK cells did not seem to be largely involved whereas macrophages might be good candidates as anti-xenograft effectors. PMID:3882112

  14. Effects of cobalt chloride on phenotypes of normal human saphenous vein smooth muscle cells

    PubMed Central

    Li, Jing; Wang, Huai-Ming

    2014-01-01

    To explore the cellular adaptations and responses to hypoxia in normal human saphenous vein smooth muscle cells (SMCs) and presume what roles phenotypic modulation of normal human saphenous vein SMCs would play in varicose vein of lower extremity, we used cobalt chloride (CoCl2), a hypoxia mimetic, to treat normal human saphenous vein SMCs in vitro. The proliferating ability of cells exposed to serial dilutions of CoCl2 (0, 200, 300, 400 and 500 μM) at 24 h, 48 h and 72 h respectively was detected by MTT assay. Wound healing assay was used to observe the migrating ability of cells under CoCl2 (200 μM) treatment for 8 days continuously. Hoechst 33258 stain was used to determine whether hypoxia induced by CoCl2 could cause apoptosis of normal human saphenous vein SMCs. We found that CoCl2 enhanced the proliferation and inhibited the migration of normal human saphenous vein SMCs. The apparent morphous of normal human saphenous vein SMCs under chronic CoCl2 treatment was significantly changed compared to no CoCl2 treated control, but this process did not relate to cell apoptosis. To conclude, our results support the concept that the phenotypes of normal human saphenous vein SMCs could be influenced by hypoxia stimulus. Cellular structural and functional changes under chronic hypoxia in normal human saphenous vein SMCs might play important roles in the development of varicose veins of lower extremity. PMID:25663990

  15. Effects of cobalt chloride on phenotypes of normal human saphenous vein smooth muscle cells.

    PubMed

    Li, Jing; Wang, Huai-Ming

    2014-01-01

    To explore the cellular adaptations and responses to hypoxia in normal human saphenous vein smooth muscle cells (SMCs) and presume what roles phenotypic modulation of normal human saphenous vein SMCs would play in varicose vein of lower extremity, we used cobalt chloride (CoCl2), a hypoxia mimetic, to treat normal human saphenous vein SMCs in vitro. The proliferating ability of cells exposed to serial dilutions of CoCl2 (0, 200, 300, 400 and 500 μM) at 24 h, 48 h and 72 h respectively was detected by MTT assay. Wound healing assay was used to observe the migrating ability of cells under CoCl2 (200 μM) treatment for 8 days continuously. Hoechst 33258 stain was used to determine whether hypoxia induced by CoCl2 could cause apoptosis of normal human saphenous vein SMCs. We found that CoCl2 enhanced the proliferation and inhibited the migration of normal human saphenous vein SMCs. The apparent morphous of normal human saphenous vein SMCs under chronic CoCl2 treatment was significantly changed compared to no CoCl2 treated control, but this process did not relate to cell apoptosis. To conclude, our results support the concept that the phenotypes of normal human saphenous vein SMCs could be influenced by hypoxia stimulus. Cellular structural and functional changes under chronic hypoxia in normal human saphenous vein SMCs might play important roles in the development of varicose veins of lower extremity. PMID:25663990

  16. Automatic concept recognition using the Human Phenotype Ontology reference and test suite corpora

    PubMed Central

    Groza, Tudor; Köhler, Sebastian; Doelken, Sandra; Collier, Nigel; Oellrich, Anika; Smedley, Damian; Couto, Francisco M; Baynam, Gareth; Zankl, Andreas; Robinson, Peter N.

    2015-01-01

    Concept recognition tools rely on the availability of textual corpora to assess their performance and enable the identification of areas for improvement. Typically, corpora are developed for specific purposes, such as gene name recognition. Gene and protein name identification are longstanding goals of biomedical text mining, and therefore a number of different corpora exist. However, phenotypes only recently became an entity of interest for specialized concept recognition systems, and hardly any annotated text is available for performance testing and training. Here, we present a unique corpus, capturing text spans from 228 abstracts manually annotated with Human Phenotype Ontology (HPO) concepts and harmonized by three curators, which can be used as a reference standard for free text annotation of human phenotypes. Furthermore, we developed a test suite for standardized concept recognition error analysis, incorporating 32 different types of test cases corresponding to 2164 HPO concepts. Finally, three established phenotype concept recognizers (NCBO Annotator, OBO Annotator and Bio-LarK CR) were comprehensively evaluated, and results are reported against both the text corpus and the test suites. The gold standard and test suites corpora are available from http://bio-lark.org/hpo_res.html. Database URL: http://bio-lark.org/hpo_res.html PMID:25725061

  17. The Human Phenotype Ontology: Semantic Unification of Common and Rare Disease

    PubMed Central

    Groza, Tudor; Köhler, Sebastian; Moldenhauer, Dawid; Vasilevsky, Nicole; Baynam, Gareth; Zemojtel, Tomasz; Schriml, Lynn Marie; Kibbe, Warren Alden; Schofield, Paul N.; Beck, Tim; Vasant, Drashtti; Brookes, Anthony J.; Zankl, Andreas; Washington, Nicole L.; Mungall, Christopher J.; Lewis, Suzanna E.; Haendel, Melissa A.; Parkinson, Helen; Robinson, Peter N.

    2015-01-01

    The Human Phenotype Ontology (HPO) is widely used in the rare disease community for differential diagnostics, phenotype-driven analysis of next-generation sequence-variation data, and translational research, but a comparable resource has not been available for common disease. Here, we have developed a concept-recognition procedure that analyzes the frequencies of HPO disease annotations as identified in over five million PubMed abstracts by employing an iterative procedure to optimize precision and recall of the identified terms. We derived disease models for 3,145 common human diseases comprising a total of 132,006 HPO annotations. The HPO now comprises over 250,000 phenotypic annotations for over 10,000 rare and common diseases and can be used for examining the phenotypic overlap among common diseases that share risk alleles, as well as between Mendelian diseases and common diseases linked by genomic location. The annotations, as well as the HPO itself, are freely available. PMID:26119816

  18. The Human Phenotype Ontology: Semantic Unification of Common and Rare Disease.

    PubMed

    Groza, Tudor; Köhler, Sebastian; Moldenhauer, Dawid; Vasilevsky, Nicole; Baynam, Gareth; Zemojtel, Tomasz; Schriml, Lynn Marie; Kibbe, Warren Alden; Schofield, Paul N; Beck, Tim; Vasant, Drashtti; Brookes, Anthony J; Zankl, Andreas; Washington, Nicole L; Mungall, Christopher J; Lewis, Suzanna E; Haendel, Melissa A; Parkinson, Helen; Robinson, Peter N

    2015-07-01

    The Human Phenotype Ontology (HPO) is widely used in the rare disease community for differential diagnostics, phenotype-driven analysis of next-generation sequence-variation data, and translational research, but a comparable resource has not been available for common disease. Here, we have developed a concept-recognition procedure that analyzes the frequencies of HPO disease annotations as identified in over five million PubMed abstracts by employing an iterative procedure to optimize precision and recall of the identified terms. We derived disease models for 3,145 common human diseases comprising a total of 132,006 HPO annotations. The HPO now comprises over 250,000 phenotypic annotations for over 10,000 rare and common diseases and can be used for examining the phenotypic overlap among common diseases that share risk alleles, as well as between Mendelian diseases and common diseases linked by genomic location. The annotations, as well as the HPO itself, are freely available. PMID:26119816

  19. Metagenomic predictions: from microbiome to complex health and environmental phenotypes in humans and cattle.

    PubMed

    Ross, Elizabeth M; Moate, Peter J; Marett, Leah C; Cocks, Ben G; Hayes, Ben J

    2013-01-01

    Mammals have a large cohort of endo- and ecto- symbiotic microorganisms (the microbiome) that potentially influence host phenotypes. There have been numerous exploratory studies of these symbiotic organisms in humans and other animals, often with the aim of relating the microbiome to a complex phenotype such as body mass index (BMI) or disease state. Here, we describe an efficient methodology for predicting complex traits from quantitative microbiome profiles. The method was demonstrated by predicting inflammatory bowel disease (IBD) status and BMI from human microbiome data, and enteric greenhouse gas production from dairy cattle rumen microbiome profiles. The method uses unassembled massively parallel sequencing (MPS) data to form metagenomic relationship matrices (analogous to genomic relationship matrices used in genomic predictions) to predict IBD, BMI and methane production phenotypes with useful accuracies (r = 0.423, 0.422 and 0.466 respectively). Our results show that microbiome profiles derived from MPS can be used to predict complex phenotypes of the host. Although the number of biological replicates used here limits the accuracy that can be achieved, preliminary results suggest this approach may surpass current prediction accuracies that are based on the host genome. This is especially likely for traits that are largely influenced by the gut microbiota, for example digestive tract disorders or metabolic functions such as enteric methane production in cattle. PMID:24023808

  20. Metagenomic Predictions: From Microbiome to Complex Health and Environmental Phenotypes in Humans and Cattle

    PubMed Central

    Ross, Elizabeth M.; Moate, Peter J.; Marett, Leah C.; Cocks, Ben G.; Hayes, Ben J.

    2013-01-01

    Mammals have a large cohort of endo- and ecto- symbiotic microorganisms (the microbiome) that potentially influence host phenotypes. There have been numerous exploratory studies of these symbiotic organisms in humans and other animals, often with the aim of relating the microbiome to a complex phenotype such as body mass index (BMI) or disease state. Here, we describe an efficient methodology for predicting complex traits from quantitative microbiome profiles. The method was demonstrated by predicting inflammatory bowel disease (IBD) status and BMI from human microbiome data, and enteric greenhouse gas production from dairy cattle rumen microbiome profiles. The method uses unassembled massively parallel sequencing (MPS) data to form metagenomic relationship matrices (analogous to genomic relationship matrices used in genomic predictions) to predict IBD, BMI and methane production phenotypes with useful accuracies (r = 0.423, 0.422 and 0.466 respectively). Our results show that microbiome profiles derived from MPS can be used to predict complex phenotypes of the host. Although the number of biological replicates used here limits the accuracy that can be achieved, preliminary results suggest this approach may surpass current prediction accuracies that are based on the host genome. This is especially likely for traits that are largely influenced by the gut microbiota, for example digestive tract disorders or metabolic functions such as enteric methane production in cattle. PMID:24023808

  1. Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells

    PubMed Central

    Reichert, Doreen; Friedrichs, Jens; Ritter, Steffi; Käubler, Theresa; Werner, Carsten; Bornhäuser, Martin; Corbeil, Denis

    2015-01-01

    Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used. PMID:26498381

  2. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  3. The development of a phantom to determine foetal organ doses from 131I in the foetal thyroid

    NASA Astrophysics Data System (ADS)

    O'Hare, N.; Murphy, D.; Malone, J. F.

    2000-09-01

    Iodine can accumulate in the foetal thyroid from the twelfth week of gestation onwards. If the iodine taken up by the foetal thyroid is in the form of 131I then the thyroid and its proximal tissues and organs will be irradiated. Several mathematical models exist in the literature on foetal/maternal iodine kinetics. However, very few studies have been performed where the foetal thyroid had been physically modelled thus allowing the determination of foetal organ dosimetry from 131I in the foetal thyroid. Here, the development of such a physical model or phantom is described and dosimetry results obtained from the phantom are discussed. The phantom is of Perspex construction, the dimensions of which are sufficient to incorporate models of the foetus at 16, 24 and 36 weeks' gestational age. The dosimetry of two organs is presented, that of the brain and the thymus. The results show that the measured absorbed dose is comparable with that calculated using modified MIRD dosimetry and traditional methods. The results also show that the dose to the thymus is greater than that of the brain by a factor of almost 30 for 16 weeks' gestational age.

  4. Evaluation of the foetal time to death in mice after application of direct and indirect euthanasia methods.

    PubMed

    Muñoz-Mediavilla, C; Cámara, J A; Salazar, S; Segui, B; Sanguino, D; Mulero, F; de la Cueva, E; Blanco, I

    2016-04-01

    Directive 2010/63/EU on the protection of animals used for scientific purposes requires that the killing of mammal foetuses during the last third of their gestational period should be accomplished through effective and humane methods. The fact that murine foetuses are resistant to hypoxia-mediated euthanasia renders the current euthanasia methods ineffective or humane for the foetuses when these methods are applied to pregnant female mice. We have assessed the time to death of foetuses after performing either indirect (dam euthanasia) or direct (via intraplacental injection - a new approach to euthanasia) euthanasia methods in order to determine a euthanasia method that is appropriate, ethical and efficient for the killing of mouse foetuses. The respective times to death of foetuses after performing the three most commonly used euthanasia methods (namely cervical dislocation, CO2inhalation and intraperitoneal sodium pentobarbital administration) were recorded. Absence of foetal heartbeat was monitored via ultrasound. We consider that the most effective and humane method of foetal euthanasia was the one able to achieve foetal death within the shortest possible period of time. Among the indirect euthanasia methods assessed, the administration of a sodium pentobarbital overdose to pregnant female mice was found to be the fastest for foetuses, with an average post-treatment foetal death of approximately 29.8 min. As for the direct euthanasia method assessed, foetal time to death after intraplacental injection of sodium pentobarbital was approximately 14 min. Significant differences among the different mouse strains employed were found. Based on the results obtained in our study, we consider that the administration of a sodium pentobarbital overdose by intraplacental injection to be an effective euthanasia method for murine foetuses. PMID:26265244

  5. Human CLPP reverts the longevity phenotype of a fungal ClpP deletion strain

    PubMed Central

    Fischer, Fabian; Weil, Andrea; Hamann, Andrea; Osiewacz, Heinz D.

    2013-01-01

    Mitochondrial maintenance crucially depends on the quality control of proteins by various chaperones, proteases and repair enzymes. While most of the involved components have been studied in some detail, little is known on the biological role of the CLPXP protease complex located in the mitochondrial matrix. Here we show that deletion of PaClpP, encoding the CLP protease proteolytic subunit CLPP, leads to an unexpected healthy phenotype and increased lifespan of the fungal ageing model organism Podospora anserina. This phenotype can be reverted by expression of human ClpP in the fungal deletion background, demonstrating functional conservation of human and fungal CLPP. Our results show that the biological role of eukaryotic CLP proteases can be studied in an experimentally accessible model organism. PMID:23360988

  6. Phenotypic robustness and the assortativity signature of human transcription factor networks.

    PubMed

    Pechenick, Dov A; Payne, Joshua L; Moore, Jason H

    2014-08-01

    Many developmental, physiological, and behavioral processes depend on the precise expression of genes in space and time. Such spatiotemporal gene expression phenotypes arise from the binding of sequence-specific transcription factors (TFs) to DNA, and from the regulation of nearby genes that such binding causes. These nearby genes may themselves encode TFs, giving rise to a transcription factor network (TFN), wherein nodes represent TFs and directed edges denote regulatory interactions between TFs. Computational studies have linked several topological properties of TFNs - such as their degree distribution - with the robustness of a TFN's gene expression phenotype to genetic and environmental perturbation. Another important topological property is assortativity, which measures the tendency of nodes with similar numbers of edges to connect. In directed networks, assortativity comprises four distinct components that collectively form an assortativity signature. We know very little about how a TFN's assortativity signature affects the robustness of its gene expression phenotype to perturbation. While recent theoretical results suggest that increasing one specific component of a TFN's assortativity signature leads to increased phenotypic robustness, the biological context of this finding is currently limited because the assortativity signatures of real-world TFNs have not been characterized. It is therefore unclear whether these earlier theoretical findings are biologically relevant. Moreover, it is not known how the other three components of the assortativity signature contribute to the phenotypic robustness of TFNs. Here, we use publicly available DNaseI-seq data to measure the assortativity signatures of genome-wide TFNs in 41 distinct human cell and tissue types. We find that all TFNs share a common assortativity signature and that this signature confers phenotypic robustness to model TFNs. Lastly, we determine the extent to which each of the four components of the

  7. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease.

    PubMed

    Benraiss, Abdellatif; Wang, Su; Herrlinger, Stephanie; Li, Xiaojie; Chandler-Militello, Devin; Mauceri, Joseph; Burm, Hayley B; Toner, Michael; Osipovitch, Mikhail; Jim Xu, Qiwu; Ding, Fengfei; Wang, Fushun; Kang, Ning; Kang, Jian; Curtin, Paul C; Brunner, Daniela; Windrem, Martha S; Munoz-Sanjuan, Ignacio; Nedergaard, Maiken; Goldman, Steven A

    2016-01-01

    The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial chimeras are hyperexcitable. Conversely, normal glia can ameliorate disease phenotype in transgenic HD mice, as striatal transplantation of normal glia rescues aspects of electrophysiological and behavioural phenotype, restores interstitial potassium homeostasis, slows disease progression and extends survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder. PMID:27273432

  8. Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

    PubMed Central

    Saxon, P J; Schultz, R A; Stanbridge, E J; Friedberg, E C

    1989-01-01

    Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15. Images Figure 3 Figure 4 PMID:2929593

  9. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease

    PubMed Central

    Benraiss, Abdellatif; Wang, Su; Herrlinger, Stephanie; Li, Xiaojie; Chandler-Militello, Devin; Mauceri, Joseph; Burm, Hayley B.; Toner, Michael; Osipovitch, Mikhail; Jim Xu, Qiwu; Ding, Fengfei; Wang, Fushun; Kang, Ning; Kang, Jian; Curtin, Paul C.; Brunner, Daniela; Windrem, Martha S.; Munoz-Sanjuan, Ignacio; Nedergaard, Maiken; Goldman, Steven A.

    2016-01-01

    The causal contribution of glial pathology to Huntington disease (HD) has not been heavily explored. To define the contribution of glia to HD, we established human HD glial chimeras by neonatally engrafting immunodeficient mice with mutant huntingtin (mHTT)-expressing human glial progenitor cells (hGPCs), derived from either human embryonic stem cells or mHTT-transduced fetal hGPCs. Here we show that mHTT glia can impart disease phenotype to normal mice, since mice engrafted intrastriatally with mHTT hGPCs exhibit worse motor performance than controls, and striatal neurons in mHTT glial chimeras are hyperexcitable. Conversely, normal glia can ameliorate disease phenotype in transgenic HD mice, as striatal transplantation of normal glia rescues aspects of electrophysiological and behavioural phenotype, restores interstitial potassium homeostasis, slows disease progression and extends survival in R6/2 HD mice. These observations suggest a causal role for glia in HD, and further suggest a cell-based strategy for disease amelioration in this disorder. PMID:27273432

  10. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    PubMed

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  11. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee

    PubMed Central

    Advani, Alexander S.; Chen, Annie Y.; Babbitt, Courtney C.

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  12. Human Endometrial Side Population Cells Exhibit Genotypic, Phenotypic and Functional Features of Somatic Stem Cells

    PubMed Central

    Cervelló, Irene; Gil-Sanchis, Claudia; Mas, Aymara; Delgado-Rosas, Francisco; Martínez-Conejero, José Antonio; Galán, Amparo; Martínez-Romero, Alicia; Martínez, Sebastian; Navarro, Ismael; Ferro, Jaime; Horcajadas, José Antonio; Esteban, Francisco José; O'Connor, José Enrique; Pellicer, Antonio; Simón, Carlos

    2010-01-01

    During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women's cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC. PMID:20585575

  13. Tissue Metabonomic Phenotyping for Diagnosis and Prognosis of Human Colorectal Cancer

    PubMed Central

    Tian, Yuan; Xu, Tangpeng; Huang, Jia; Zhang, Limin; Xu, Shan; Xiong, Bin; Wang, Yulan; Tang, Huiru

    2016-01-01

    Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide and prognosis based on the conventional histological grading method for CRC remains poor. To better the situation, we analyzed the metabonomic signatures of 50 human CRC tissues and their adjacent non-involved tissues (ANIT) using high-resolution magic-angle spinning (HRMAS) 1H NMR spectroscopy together with the fatty acid compositions of these tissues using GC-FID/MS. We showed that tissue metabolic phenotypes not only discriminated CRC tissues from ANIT, but also distinguished low-grade tumor tissues (stages I-II) from the high-grade ones (stages III-IV) with high sensitivity and specificity in both cases. Metabonomic phenotypes of CRC tissues differed significantly from that of ANIT in energy metabolism, membrane biosynthesis and degradations, osmotic regulations together with the metabolism of proteins and nucleotides. Amongst all CRC tissues, the stage I tumors exhibited largest differentiations from ANIT. The combination of the differentiating metabolites showed outstanding collective power for differentiating cancer from ANIT and for distinguishing CRC tissues at different stages. These findings revealed details in the typical metabonomic phenotypes associated with CRC tissues nondestructively and demonstrated tissue metabonomic phenotyping as an important molecular pathology tool for diagnosis and prognosis of cancerous solid tumors. PMID:26876567

  14. Nanopatterned Human iPSC-based Model of a Dystrophin-Null Cardiomyopathic Phenotype

    PubMed Central

    Macadangdang, Jesse; Guan, Xuan; Smith, Alec S.T.; Lucero, Rachel; Czerniecki, Stefan; Childers, Martin K.; Mack, David L.; Kim, Deok-Ho

    2015-01-01

    Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer unprecedented opportunities to study inherited heart conditions in vitro, but are phenotypically immature, limiting their ability to effectively model adult-onset diseases. Cardiomyopathy is becoming the leading cause of death in patients with Duchenne muscular dystrophy (DMD), but the pathogenesis of this disease phenotype is not fully understood. Therefore, we aimed to test whether biomimetic nanotopography could further stratify the disease phenotype of DMD hiPSC-CMs to create more translationally relevant cardiomyocytes for disease modeling applications. We found that anisotropic nanotopography was necessary to distinguish structural differences between normal and DMD hiPSC-CMs, as these differences were masked on conventional flat substrates. DMD hiPSC-CMs exhibited a diminished structural and functional response to the underlying nanotopography compared to normal cardiomyocytes at both the macroscopic and subcellular levels. This blunted response may be due to a lower level of actin cytoskeleton turnover as measured by fluorescence recovery after photobleaching. Taken together these data suggest that DMD hiPSC-CMs are less adaptable to changes in their extracellular environment, and highlight the utility of nanotopographic substrates for effectively stratifying normal and structural cardiac disease phenotypes in vitro. PMID:26366230

  15. Combining Human Disease Genetics and Mouse Model Phenotypes towards Drug Repositioning for Parkinson’s disease

    PubMed Central

    Chen, Yang; Cai, Xiaoshu; Xu, Rong

    2015-01-01

    Parkinson’s disease (PD) is a severe neurodegenerative disorder without effective treatments. Here, we present a novel drug repositioning approach to predict new drugs for PD leveraging both disease genetics and large amounts of mouse model phenotypes. First, we identified PD-specific mouse phenotypes using well-studied human disease genes. Then we searched all FDA-approved drugs for candidates that share similar mouse phenotype profiles with PD. We demonstrated the validity of our approach using drugs that have been approved for PD: 10 approved PD drugs were ranked within top 10% among 1197 candidates. In predicting novel PD drugs, our approach achieved a mean average precision of 0.24, which is significantly higher (pphenotype data. Comparison of gene expression profiles between PD and top-ranked drug candidates indicates that quetiapine has the potential to treat PD. PMID:26958284

  16. Gax regulates human vascular smooth muscle cell phenotypic modulation and vascular remodeling

    PubMed Central

    Zheng, Hui; Hu, Zhenlei; Zhai, Xinming; Wang, Yongyi; Liu, Jidong; Wang, Weijun; Xue, Song

    2016-01-01

    Abnormal phenotypic modulation of vascular smooth muscle cells (VSMCs) is a hallmark of cardiovascular diseases such as atherosclerosis, hypertension and restenosis after angioplasty. Transcription factors have emerged as critical regulators for VSMCs function, and recently we verified inhibiting transcription factor Gax was important for controlling VSMCs proliferation and migration. This study aimed to determine its role in phenotypic modulation of VSMCs. Western blot revealed that overexpression of Gax increased expression of VSMCs differentiation marker genes such as calponin and SM-MHC 11. Then, Gax overexpression potently suppressed proliferation and migration of VSMCs with or without platelet-derived growth factor-induced-BB (PDGF-BB) stimuli whereas Gax silencing inhibited these processes. Furthermore, cDNA array analysis indicated that Rap1A gene was the downstream target of Gax in human VSMCs. And overexpression of Gax significantly inhibited expression of Rap1A in VSMCs with or without PDGF-BB stimuli. Moreover, overexpression of Rap1A decreased expression of VSMCs differentiation marker genes and increased proliferation and migration of VSMCs with or without PDGF-BB stimuli. Finally, Gax overexpression significantly inhibited the neointimal formation in carotid artery injury of mouse models, specifically through maintaining VSMCs contractile phenotype by decreasing Rap1A expression. In conclusion, these results indicated that Gax was a regulator of human VSMCs phenotypic modulation by targeting Rap1A gene, which suggested that targeting Gax or its downstream targets in human VSMCs may provide an attractive approach for the prevention and treatment of cardiovascular diseases. PMID:27508012

  17. ClinVar: public archive of relationships among sequence variation and human phenotype

    PubMed Central

    Landrum, Melissa J.; Lee, Jennifer M.; Riley, George R.; Jang, Wonhee; Rubinstein, Wendy S.; Church, Deanna M.; Maglott, Donna R.

    2014-01-01

    ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/) provides a freely available archive of reports of relationships among medically important variants and phenotypes. ClinVar accessions submissions reporting human variation, interpretations of the relationship of that variation to human health and the evidence supporting each interpretation. The database is tightly coupled with dbSNP and dbVar, which maintain information about the location of variation on human assemblies. ClinVar is also based on the phenotypic descriptions maintained in MedGen (http://www.ncbi.nlm.nih.gov/medgen). Each ClinVar record represents the submitter, the variation and the phenotype, i.e. the unit that is assigned an accession of the format SCV000000000.0. The submitter can update the submission at any time, in which case a new version is assigned. To facilitate evaluation of the medical importance of each variant, ClinVar aggregates submissions with the same variation/phenotype combination, adds value from other NCBI databases, assigns a distinct accession of the format RCV000000000.0 and reports if there are conflicting clinical interpretations. Data in ClinVar are available in multiple formats, including html, download as XML, VCF or tab-delimited subsets. Data from ClinVar are provided as annotation tracks on genomic RefSeqs and are used in tools such as Variation Reporter (http://www.ncbi.nlm.nih.gov/variation/tools/reporter), which reports what is known about variation based on user-supplied locations. PMID:24234437

  18. Urban particle-induced apoptosis and phenotype shifts in human alveolar macrophages.

    PubMed Central

    Holian, A; Hamilton, R F; Morandi, M T; Brown, S D; Li, L

    1998-01-01

    Epidemiological studies report a small but positive association between short-term increases in airborne particulate matter and small increases in morbidity and mortality from respiratory and cardiovascular disease in urban areas. However, the lack of a mechanistic explanation to link particle exposure and human health effects makes it difficult to validate the human health effects. The present study tested the hypothesis that urban particles could cause apoptosis of human alveolar macrophages(AM) and a shift of their phenotypes to a higher immune active state, which would provide a mechanism to explain an inflammatory response. Freshly isolated human AM were incubated for 24 hr with urban particles (#1648 and #1649), Mount Saint Helen's ash (MSH), and residual oil fly ash (ROFA).Cell viability was assessed by trypan blue exclusion and apoptosis was demonstrated by morphology, cell death ELISA, and DNA ladder formation. Additionally, AM were characterized according to RFD1(+) (immune stimulatory macrophages) and RFD1(+)7(+) (suppressor macrophages) phenotypes by flow cytometry. ROFA particles caused AM necrosis at concentrations as low as 10 microg/ml, urban particles had no effect except at 200 microg/ml, and MSH had no effect at 200 microg/ml. ROFA (25 microg/ml) and particles #1648 or #1649 (100 microg/ml) caused apoptosis of AM by all three criteria, but 200 microg/ml MSH had no effect. Finally, 25 microg/ml ROFA and 100 microg/ml particles #1648 or #1649 up regulated the expression of the RFD1(+) AM phenotype, while only ROFA decreased the RFD1(+)7(+) phenotype. Consequently, ROFA and urban particles can induce apoptosis of human AM and increase the ratio of AM phenotypes toward a higher immune active state (i.e., increased RFD1(+):RFD1(+)7(+) ratio). Ifurban particles cause similar changes in vivo, this could result in lung inflammation and possible increased pulmonary and cardiovascular disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID

  19. The human gene map for performance and health-related fitness phenotypes: The 2006-2007 update

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This update of the human gene map for physical performance and health-related fitness phenotypes covers the research advances reported in 2006 and 2007. The genes and markers with evidence of association or linkage with a performance or a fitness phenotype in sedentary or active people, in responses...

  20. Human Peripheral Clocks: Applications for Studying Circadian Phenotypes in Physiology and Pathophysiology

    PubMed Central

    Saini, Camille; Brown, Steven A.; Dibner, Charna

    2015-01-01

    Most light-sensitive organisms on earth have acquired an internal system of circadian clocks allowing the anticipation of light or darkness. In humans, the circadian system governs nearly all aspects of physiology and behavior. Circadian phenotypes, including chronotype, vary dramatically among individuals and over individual lifespan. Recent studies have revealed that the characteristics of human skin fibroblast clocks correlate with donor chronotype. Given the complexity of circadian phenotype assessment in humans, the opportunity to study oscillator properties by using cultured primary cells has the potential to uncover molecular details difficult to assess directly in humans. Since altered properties of the circadian oscillator have been associated with many diseases including metabolic disorders and cancer, clock characteristics assessed in additional primary cell types using similar technologies might represent an important tool for exploring the connection between chronotype and disease, and for diagnostic purposes. Here, we review implications of this approach for gathering insights into human circadian rhythms and their function in health and disease. PMID:26029154

  1. Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes

    PubMed Central

    Boisson-Dupuis, Stephanie; Kong, Xiao-Fei; Okada, Satoshi; Cypowyj, Sophie; Puel, Anne; Abel, Laurent; Casanova, Jean-Laurent

    2012-01-01

    The genetic dissection of various human infectious diseases has led to the definition of inborn errors of human STAT1 immunity of four types, including (i) autosomal recessive (AR) complete STAT1 deficiency, (ii) AR partial STAT1 deficiency, (iii) autosomal dominant (AD) STAT1 deficiency, and (iv) AD gain of STAT1 activity. The two types of AR STAT1 defect give rise to a broad infectious phenotype with susceptibility to intramacrophagic bacteria (mostly mycobacteria) and viruses (herpes viruses at least), due principally to the impairment of IFN-γ-mediated and IFN-α/β-mediated immunity, respectively. Clinical outcome depends on the extent to which the STAT1 defect decreases responsiveness to these cytokines. AD STAT1 deficiency selectively predisposes individuals to mycobacterial disease, owing to the impairment of IFN-γ-mediated immunity, as IFN-α/β-mediated immunity is maintained. Finally, AD gain of STAT1 activity is associated with autoimmunity, probably owing to an enhancement of IFN-α/β-mediated immunity. More surprisingly, it is also associated with chronic mucocutaneous candidiasis, through as yet undetermined mechanisms involving an inhibition of the development of IL-17-producing T cells. Thus, germline mutations in human STAT1 define four distinct clinical disorders. Various combinations of viral, mycobacterial and fungal infections are therefore allelic at the human STAT1 locus. These experiments of Nature neatly highlight the clinical and immunological impact of the human genetic dissection of infectious phenotypes. PMID:22651901

  2. A PC-aided optical foetal heart rate detection system.

    PubMed

    Oweis, Rami J; As'ad, Hala; Aldarawsheh, Amany; Al-Khdeirat, Rawan; Lwissy, Kaldoun

    2014-01-01

    Safe monitoring of foetal heart rate is a valuable tool for the healthy evolution and wellbeing of both foetus and mother. This paper presents a non-invasive optical technique that allows for foetal heart rate detection using a photovoltaic infrared (IR) detector placed on the mother's abdomen. The system presented here consists of a photoplethysmography (PPG) circuit, abdomen circuit and a personal computer equipped with MATLAB. A near IR beam having a wavelength of 880 nm is transmitted through the mother's abdomen and foetal tissue. The received abdominal signal that conveys information pertaining to the mother and foetal heart rate is sensed by a low noise photodetector. The PC receives the signal through the National Instrumentation Data Acquisition Card (NIDAQ). After synchronous detection of the abdominal and finger PPG signals, the designed MATLAB-based software saves, analyses and extracts information related to the foetal heart rate. Extraction is carried out using recursive least squares adaptive filtration. Measurements on eight pregnant women with gestational periods ranging from 35-39 weeks were performed using the proposed system and CTG. Results show a correlation coefficient of 0.978 and a correlation confidence interval between 88-99.6%. The t test results in a p value of 0.034, which is less than 0.05. Low power, low cost, high signal-to-noise ratio, reduction of ambient light effect and ease of use are the main characteristics of the proposed system. PMID:24195701

  3. Human CYP1A1 gene: cosegregation of the enzyme inducibility phenotype and an RFLP.

    PubMed Central

    Petersen, D D; McKinney, C E; Ikeya, K; Smith, H H; Bale, A E; McBride, O W; Nebert, D W

    1991-01-01

    The human CYP1A1 (cytochrome P1450) gene encodes an enzyme involved in the activation of procarcinogens, such as benzo[a]pyrene, to the ultimate reactive intermediate. Approximately 10% of the human population exhibit high CYP1A1 inducibility, and Kouri et al. reported that the high-inducibility phenotype might be at greater risk than low-inducibility individuals for cigarette smoke-induced bronchogenic carcinoma. In one 3-generation family of 15 individuals, we show here that the high-CYP1A1-inducibility phenotype segregates concordantly with an infrequent polymorphic site located 450 bases downstream from the CYP1A1 gene. Our findings are consistent with the study of Kawajiri et al., who demonstrated an association between this polymorphism and an increased incidence of squamous-cell lung cancer. Our data suggest that the CYP1A1 structural gene, or a region near this gene, might be correlated with the inducibility phenotype. Images Figure 3 PMID:1707592

  4. The perivascular phenotype and behaviors of dedifferentiated cells derived from human mature adipocytes.

    PubMed

    Song, Ning; Kou, Liang; Lu, Xiao-Wen; Sugawara, Atsunori; Shimizu, Yutaka; Wu, Min-Ke; Du, Li; Wang, Hang; Sato, Soh; Shen, Jie-Fei

    2015-02-13

    Derived from mature adipocytes, dedifferentiated fat (DFAT) cells represent a special group of multipotent cells. However, their phenotype and cellular nature remain unclear. Our study found that human DFAT cells adopted perivascular characteristics and behaviors. Flow cytometry and immunofluorescent staining revealed that human DFAT cells positively expressed markers highly related to perivascular cell lineages, such as CD140b, NG2 and desmin, but were negative for common endothelial markers, including CD31, CD34, and CD309. Furthermore, DFAT cells displayed vascular network formation ability in Matrigel, and they noticeably promoted and stabilized the vessel structures formed by human umbilical vascular endothelial cells (HUVECs) in vitro. These results provide novel evidence on the pericyte nature of human DFAT cells, further supporting the recent model for the perivascular origin of adult stem cells, in which tissue-specific progenitor cells in mesenchymal tissues associate with blood vessels, exhibiting perivascular characteristics and functions. PMID:25603054

  5. Human memory T cells with a naive phenotype accumulate with aging and respond to persistent viruses.

    PubMed

    Pulko, Vesna; Davies, John S; Martinez, Carmine; Lanteri, Marion C; Busch, Michael P; Diamond, Michael S; Knox, Kenneth; Bush, Erin C; Sims, Peter A; Sinari, Shripad; Billheimer, Dean; Haddad, Elias K; Murray, Kristy O; Wertheimer, Anne M; Nikolich-Žugich, Janko

    2016-08-01

    The number of naive T cells decreases and susceptibility to new microbial infections increases with age. Here we describe a previously unknown subset of phenotypically naive human CD8(+) T cells that rapidly secreted multiple cytokines in response to persistent viral antigens but differed transcriptionally from memory and effector T cells. The frequency of these CD8(+) T cells, called 'memory T cells with a naive phenotype' (TMNP cells), increased with age and after severe acute infection and inversely correlated with the residual capacity of the immune system to respond to new infections with age. CD8(+) TMNP cells represent a potential new target for the immunotherapy of persistent infections and should be accounted for and subtracted from the naive pool if truly naive T cells are needed to respond to antigens. PMID:27270402

  6. Hepatic Localization of Macrophage Phenotypes during Fibrogenesis and Resolution of Fibrosis in Mice and Humans

    PubMed Central

    Beljaars, Leonie; Schippers, Marlies; Reker-Smit, Catharina; Martinez, Fernando O.; Helming, Laura; Poelstra, Klaas; Melgert, Barbro N.

    2014-01-01

    Macrophages have been found to both promote liver fibrosis and contribute to its resolution by acquiring different phenotypes based on signals from the micro-environment. The best-characterized phenotypes in the macrophage spectrum are labeled M1 (classically activated) and M2 (alternatively activated). Until now the in situ localization of these phenotypes in diseased livers is poorly described. In this study, we therefore aimed to localize and quantify M1- and M2-dominant macrophages in diseased mouse and human livers. The scarred collagen-rich areas in cirrhotic human livers and in CCl4-damaged mouse livers contained many macrophages. Though total numbers of macrophages were higher in fibrotic livers, the number of parenchymal CD68-positive macrophages was significantly lower as compared to normal. Scar-associated macrophages were further characterized as either M1-dominant (IRF-5 and interleukin-12) or M2-dominant (CD206, transglutaminase-2, and YM-1) and significantly higher numbers of both of these were detected in diseased livers as compared to healthy human and mouse livers. Interestingly, in mouse, livers undergoing resolution of fibrosis, the total number of CD68+ macrophages was significantly lower compared to their fibrotic counterparts. M2-dominant (YM-1) macrophages were almost completely gone in livers undergoing resolution, while numbers of M1-dominant (IRF-5) macrophages were almost unchanged and the proteolytic activity (MMP9) increased. In conclusion, this study shows the distribution of macrophage subsets in livers of both human and murine origin. The presence of M1- and M2-dominant macrophages side by side in fibrotic lesions suggests that both are involved in fibrotic responses, while the persistence of M1-dominant macrophages during resolution may indicate their importance in regression of fibrosis. This study emphasizes that immunohistochemical detection of M1/M2-dominant macrophages provides valuable information in addition to widely used

  7. Blue eyes in lemurs and humans: same phenotype, different genetic mechanism.

    PubMed

    Bradley, Brenda J; Pedersen, Anja; Mundy, Nicholas I

    2009-06-01

    Almost all mammals have brown or darkly-pigmented eyes (irises), but among primates, there are some prominent blue-eyed exceptions. The blue eyes of some humans and lemurs are a striking example of convergent evolution of a rare phenotype on distant branches of the primate tree. Recent work on humans indicates that blue eye color is associated with, and likely caused by, a single nucleotide polymorphism (rs12913832) in an intron of the gene HERC2, which likely regulates expression of the neighboring pigmentation gene OCA2. This raises the immediate question of whether blue eyes in lemurs might have a similar genetic basis. We addressed this by sequencing the homologous genetic region in the blue-eyed black lemur (Eulemur macaco flavifrons; N = 4) and the closely-related black lemur (Eulemur macaco macaco; N = 4), which has brown eyes. We then compared a 166-bp segment corresponding to and flanking the human eye-color-associated region in these lemurs, as well as other primates (human, chimpanzee, orangutan, macaque, ring-tailed lemur, mouse lemur). Aligned sequences indicated that this region is strongly conserved in both Eulemur macaco subspecies as well as the other primates (except blue-eyed humans). Therefore, it is unlikely that this regulatory segment plays a major role in eye color differences among lemurs as it does in humans. Although convergent phenotypes can sometimes come about via the same or similar genetic changes occurring independently, this does not seem to be the case here, as we have shown that the genetic basis of blue eyes in lemurs differs from that of humans. PMID:19278018

  8. Suppression of the chemically transformed phenotype of BHK cells by a human cDNA

    SciTech Connect

    Eiden, M.V.; MacArthur, L.; Okayama, Hiroto )

    1991-10-01

    Transformation of baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis. A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

  9. BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells.

    PubMed

    Li, Xianwu; Yang, Hsueh-Ying; Giachelli, Cecilia M

    2008-08-01

    Vascular calcification is associated with increased risk of cardiovascular events that are the most common cause of death in patients with end-stage renal disease. Clinical and experimental studies indicate that hyperphosphatemia is a risk factor for vascular calcification and cardiovascular mortality in these patients. Our previous studies demonstrated that phosphate transport through the type III sodium-dependent phosphate cotransporter, Pit-1, was necessary for phosphate-induced calcification and osteochondrogenic phenotypic change in cultured human smooth muscle cells (SMC). BMP-2 is a potent osteogenic protein required for osteoblast differentiation and bone formation that has been implicated in vascular calcification. In the present study, we have examined the effects of BMP-2 on human SMC calcification in vitro. We found that treatment of SMC with BMP-2 enhanced elevated phosphate-induced calcification, but did not induce calcification under normal phosphate conditions. mRNAs for BMP receptors, including ALK2, ALK3, ALK6, BMPR-II, ActR-IIA and ActR-IIB were all detected in human SMCs. Mechanistically, BMP-2 dose-dependently stimulated phosphate uptake in SMC (200 ng/ml BMP-2 vs. vehicle: 13.94 vs. 7.09 nmol/30 min/mg protein, respectively). Real-time PCR and Western blot revealed the upregulation of Pit-1 mRNA and protein levels, respectively, by BMP-2. More importantly, inhibition of phosphate uptake by a competitive inhibitor of sodium-dependent phosphate cotransport, phosphonoformic acid, abrogated BMP-2-induced calcification. These results indicate that phosphate transport via Pit-1 is crucial in BMP-2-regulated SMC calcification. In addition, BMP-2-induced Runx2 and inhibited SM22 expression, indicating that it promotes osteogenic phenotype transition in these cells. Thus, BMP-2 may promote vascular calcification via increased phosphate uptake and induction of osteogenic phenotype modulation in SMC. PMID:18179800

  10. Rasd2 Modulates Prefronto-Striatal Phenotypes in Humans and 'Schizophrenia-Like Behaviors' in Mice.

    PubMed

    Vitucci, Daniela; Di Giorgio, Annabella; Napolitano, Francesco; Pelosi, Barbara; Blasi, Giuseppe; Errico, Francesco; Attrotto, Maria Teresa; Gelao, Barbara; Fazio, Leonardo; Taurisano, Paolo; Di Maio, Anna; Marsili, Valentina; Pasqualetti, Massimo; Bertolino, Alessandro; Usiello, Alessandro

    2016-02-01

    Rasd2 is a thyroid hormone target gene, which encodes for a GTP-binding protein enriched in the striatum where, among other functions, it modulates dopaminergic neurotransmission. Here we report that human RASD2 mRNA is abundant in putamen, but it also occurs in the cerebral cortex, with a distinctive expression pattern that differs from that present in rodents. Consistent with its localization, we found that a genetic variation in RASD2 (rs6518956) affects postmortem prefrontal mRNA expression in healthy humans and is associated with phenotypes of relevance to schizophrenia, including prefrontal and striatal grey matter volume and physiology during working memory, as measured with magnetic resonance imaging. Interestingly, quantitative real-time PCR analysis indicated that RASD2 mRNA is slightly reduced in postmortem prefrontal cortex of patients with schizophrenia. In the attempt to uncover the neurobiological substrates associated with Rasd2 activity, we used knockout mice to analyze the in vivo influence of this G-protein on the prepulse inhibition of the startle response and psychotomimetic drug-related behavioral response. Data showed that Rasd2 mutants display deficits in basal prepulse inhibition that, in turn, exacerbate gating disruption under psychotomimetic drug challenge. Furthermore, we documented that lack of Rasd2 strikingly enhances the behavioral sensitivity to motor stimulation elicited by amphetamine and phencyclidine. Based on animal model data, along with the finding that RASD2 influences prefronto-striatal phenotypes in healthy humans, we suggest that genetic mutation or reduced levels of this G-protein might have a role in cerebral circuitry dysfunction underpinning exaggerated psychotomimetic drugs responses and development of specific biological phenotypes linked to schizophrenia. PMID:26228524

  11. Humans display a reduced set of consistent behavioral phenotypes in dyadic games

    PubMed Central

    Poncela-Casasnovas, Julia; Gutiérrez-Roig, Mario; Gracia-Lázaro, Carlos; Vicens, Julian; Gómez-Gardeñes, Jesús; Perelló, Josep; Moreno, Yamir; Duch, Jordi; Sánchez, Angel

    2016-01-01

    Socially relevant situations that involve strategic interactions are widespread among animals and humans alike. To study these situations, theoretical and experimental research has adopted a game theoretical perspective, generating valuable insights about human behavior. However, most of the results reported so far have been obtained from a population perspective and considered one specific conflicting situation at a time. This makes it difficult to extract conclusions about the consistency of individuals’ behavior when facing different situations and to define a comprehensive classification of the strategies underlying the observed behaviors. We present the results of a lab-in-the-field experiment in which subjects face four different dyadic games, with the aim of establishing general behavioral rules dictating individuals’ actions. By analyzing our data with an unsupervised clustering algorithm, we find that all the subjects conform, with a large degree of consistency, to a limited number of behavioral phenotypes (envious, optimist, pessimist, and trustful), with only a small fraction of undefined subjects. We also discuss the possible connections to existing interpretations based on a priori theoretical approaches. Our findings provide a relevant contribution to the experimental and theoretical efforts toward the identification of basic behavioral phenotypes in a wider set of contexts without aprioristic assumptions regarding the rules or strategies behind actions. From this perspective, our work contributes to a fact-based approach to the study of human behavior in strategic situations, which could be applied to simulating societies, policy-making scenario building, and even a variety of business applications. PMID:27532047

  12. Humans display a reduced set of consistent behavioral phenotypes in dyadic games.

    PubMed

    Poncela-Casasnovas, Julia; Gutiérrez-Roig, Mario; Gracia-Lázaro, Carlos; Vicens, Julian; Gómez-Gardeñes, Jesús; Perelló, Josep; Moreno, Yamir; Duch, Jordi; Sánchez, Angel

    2016-08-01

    Socially relevant situations that involve strategic interactions are widespread among animals and humans alike. To study these situations, theoretical and experimental research has adopted a game theoretical perspective, generating valuable insights about human behavior. However, most of the results reported so far have been obtained from a population perspective and considered one specific conflicting situation at a time. This makes it difficult to extract conclusions about the consistency of individuals' behavior when facing different situations and to define a comprehensive classification of the strategies underlying the observed behaviors. We present the results of a lab-in-the-field experiment in which subjects face four different dyadic games, with the aim of establishing general behavioral rules dictating individuals' actions. By analyzing our data with an unsupervised clustering algorithm, we find that all the subjects conform, with a large degree of consistency, to a limited number of behavioral phenotypes (envious, optimist, pessimist, and trustful), with only a small fraction of undefined subjects. We also discuss the possible connections to existing interpretations based on a priori theoretical approaches. Our findings provide a relevant contribution to the experimental and theoretical efforts toward the identification of basic behavioral phenotypes in a wider set of contexts without aprioristic assumptions regarding the rules or strategies behind actions. From this perspective, our work contributes to a fact-based approach to the study of human behavior in strategic situations, which could be applied to simulating societies, policy-making scenario building, and even a variety of business applications. PMID:27532047

  13. A Human Thrifty Phenotype Associated With Less Weight Loss During Caloric Restriction

    PubMed Central

    Thearle, Marie S.; Ibrahim, Mostafa; Hohenadel, Maximilian G.; Bogardus, Clifton; Krakoff, Jonathan; Votruba, Susanne B.

    2015-01-01

    Successful weight loss is variable for reasons not fully elucidated. Whether effective weight loss results from smaller reductions in energy expenditure during caloric restriction is not known. We analyzed whether obese individuals with a “thrifty” phenotype, that is, greater reductions in 24-h energy expenditure during fasting and smaller increases with overfeeding, lose less weight during caloric restriction than those with a “spendthrift” phenotype. During a weight-maintaining period, 24-h energy expenditure responses to fasting and 200% overfeeding were measured in a whole-room indirect calorimeter. Volunteers then underwent 6 weeks of 50% caloric restriction. We calculated the daily energy deficit (kilocalories per day) during caloric restriction, incorporating energy intake and waste, energy expenditure, and daily activity. We found that a smaller reduction in 24-h energy expenditure during fasting and a larger response to overfeeding predicted more weight loss over 6 weeks, even after accounting for age, sex, race, and baseline weight, as well as a greater rate of energy deficit accumulation. The success of dietary weight loss efforts is influenced by the energy expenditure response to caloric restriction. Greater decreases in energy expenditure during caloric restriction predict less weight loss, indicating the presence of thrifty and spendthrift phenotypes in obese humans. PMID:25964395

  14. Forensic DNA Phenotyping: Predicting human appearance from crime scene material for investigative purposes.

    PubMed

    Kayser, Manfred

    2015-09-01

    Forensic DNA Phenotyping refers to the prediction of appearance traits of unknown sample donors, or unknown deceased (missing) persons, directly from biological materials found at the scene. "Biological witness" outcomes of Forensic DNA Phenotyping can provide investigative leads to trace unknown persons, who are unidentifiable with current comparative DNA profiling. This intelligence application of DNA marks a substantially different forensic use of genetic material rather than that of current DNA profiling presented in the courtroom. Currently, group-specific pigmentation traits are already predictable from DNA with reasonably high accuracies, while several other externally visible characteristics are under genetic investigation. Until individual-specific appearance becomes accurately predictable from DNA, conventional DNA profiling needs to be performed subsequent to appearance DNA prediction. Notably, and where Forensic DNA Phenotyping shows great promise, this is on a (much) smaller group of potential suspects, who match the appearance characteristics DNA-predicted from the crime scene stain or from the deceased person's remains. Provided sufficient funding being made available, future research to better understand the genetic basis of human appearance will expectedly lead to a substantially more detailed description of an unknown person's appearance from DNA, delivering increased value for police investigations in criminal and missing person cases involving unknowns. PMID:25716572

  15. Esterase phenotyping in human liver in vitro: specificity of carboxylesterase inhibitors.

    PubMed

    Umehara, Ken-Ichi; Zollinger, Markus; Kigondu, Elizabeth; Witschi, Marc; Juif, Claire; Huth, Felix; Schiller, Hilmar; Chibale, Kelly; Camenisch, Gian

    2016-10-01

    1. Esterases may play a major role in the clearance of drugs with functional groups amenable to hydrolysis, particularly in the case of ester prodrugs. To understand the processes involved in the elimination of such drugs, it is necessary to determine the esterases involved. However, the tools currently available for this enzyme phenotyping are relatively scarce. 2. The work was aimed at summarizing the selectivity of esterase inhibitors for carboxylesterases 1 and 2 (CES1 and CES2) in the human liver to clarify their suitability for esterase phenotyping. Eserine, at around 10 μM, was found to be a highly specific CES2 inhibitor, whereas other esterase inhibitors turned out less selective. When used together with tacrine, which inhibits cholinesterases but not CES, and ethylenediaminetetraacetic acid (inhibitor of paraoxonases), the involvement of the hydrolyzing esterases in the hepatic clearance of a drug can be elucidated. 3. The second approach to esterase phenotyping is based on data from recombinant or isolated esterases, together with relative activity factors, which relate their activities to those of the same enzymes in subcellular fractions. 4. These two approaches will help to characterize the hydrolytic metabolism of drug candidates in a similar manner as practiced routinely for the oxidative metabolism by cytochrome P450 enzymes. PMID:26887925

  16. Le syndrome d’alcoolisme foetal

    PubMed Central

    2002-01-01

    L’alcool est un tératogène physique et comportemental. Le syndrome d’alcoolisme foetal (SAF) est un trouble courant mais encore sous-diagnostiqué découlant de la consommation d’alcool par la mère pendant la grossesse. Bien qu’il puisse être prévenu, le SAF est également invalidant. Même si le SAF est présent dans tous les groupes socioéconomiques du Canada, sa prévalence est élevée dans certaines communautés inuites et des Premières nations du Canada. Le présent énoncé porte sur la prévention, le diagnostic, le dépistage précoce et la prise en charge du SAF par les professionnels de la santé. La prévention du SAF doit s’effectuer à deux échelons. La prévention primaire consiste à éliminer le SAF par une formation en classe ou dans la collectivité et à inciter les femmes à éviter de consommer de l’alcool avant la conception et pendant la grossesse. La prévention secondaire consiste à repérer les femmes qui boivent pendant leur grossesse et à réduire leur consommation. Le présent énoncé décrit plusieurs stratégies de dépistage, dont la stratégie T-ACE (tolérance-agacement, réduction, éveil). Les dispensateurs de soins devraient recommander l’abstinence dès la première visite prénatale. Un envoi rapide en consultation en vue de traiter l’alcoolisme est recommandé pour les femmes enceintes incapables d’arrêter de boire. Le présent énoncé décrit le diagnostic de SAF, de SAF partiel ou atypique, d’anomalies congénitales et de troubles neurodéveloppementaux reliés à l’alcool. En cas d’exposition à l’alcool in utero, un diagnostic de SAF devrait être envisagé en présence d’un retard de croissance courant ou antérieur, de certaines anomalies faciales touchant la lèvre supérieure et les yeux et d’anomalies neurodéveloppementales. Ces caractéristiques sont mieux quantifiées au moyen d’une méthode diagnostique à quatre chiffres. Des stratégies de dépistage précoce des

  17. Genetic architecture for human aggression: A study of gene-phenotype relationship in OMIM.

    PubMed

    Zhang-James, Yanli; Faraone, Stephen V

    2016-07-01

    Genetic studies of human aggression have mainly focused on known candidate genes and pathways regulating serotonin and dopamine signaling and hormonal functions. These studies have taught us much about the genetics of human aggression, but no genetic locus has yet achieved genome-significance. We here present a review based on a paradoxical hypothesis that studies of rare, functional genetic variations can lead to a better understanding of the molecular mechanisms underlying complex multifactorial disorders such as aggression. We examined all aggression phenotypes catalogued in Online Mendelian Inheritance in Man (OMIM), an Online Catalog of Human Genes and Genetic Disorders. We identified 95 human disorders that have documented aggressive symptoms in at least one individual with a well-defined genetic variant. Altogether, we retrieved 86 causal genes. Although most of these genes had not been implicated in human aggression by previous studies, the most significantly enriched canonical pathways had been previously implicated in aggression (e.g., serotonin and dopamine signaling). Our findings provide strong evidence to support the causal role of these pathways in the pathogenesis of aggression. In addition, the novel genes and pathways we identified suggest additional mechanisms underlying the origins of human aggression. Genome-wide association studies with very large samples will be needed to determine if common variants in these genes are risk factors for aggression. © 2015 Wiley Periodicals, Inc. PMID:26288127

  18. Let-7b Inhibits Human Cancer Phenotype by Targeting Cytochrome P450 Epoxygenase 2J2

    PubMed Central

    Yang, Shenglan; Gong, Wei; Wang, Yan; Cianflone, Katherine; Tang, Jiarong; Wang, Dao Wen

    2012-01-01

    Background MicroRNAs (miRNAs) are small, noncoding RNA molecules of 20 to 22 nucleotides that regulate gene expression by binding to their 3′ untranslated region (3′UTR). Increasing data implicate altered miRNA participation in the progress of cancer. We previously reported that CYP2J2 epoxygenase promotes human cancer phenotypes. But whether and how CYP2J2 is regulated by miRNA is not understood. Methods and Results Using bioinformatics analysis, we found potential target sites for miRNA let-7b in 3′UTR of human CYP2J2. Luciferase and western blot assays revealed that CYP2J2 was regulated by let-7b. In addition, let-7b decreased the enzymatic activity of endogenous CYP2J2. Furthermore, let-7b may diminish cell proliferation and promote cell apoptosis of tumor cells via posttranscriptional repression of CYP2J2. Tumor xenografts were induced in nude mice by subcutaneous injection of MDA-MB-435 cells. The let-7b expression vector, pSilencer-let-7b, was injected through tail vein every 3 weeks. Let-7b significantly inhibited the tumor phenotype by targeting CYP2J2. Moreover, quantitative real-time polymerase chain reaction and western blotting were used to determine the expression levels of let-7b and CYP2J2 protein from 18 matched lung squamous cell cancer and adjacent normal lung tissues; the expression level of CYP2J2 was inversely proportional to that of let-7b. Conclusions Our results demonstrated that the decreased expression of let-7b could lead to the high expression of CYP2J2 protein in cancerous tissues. These findings suggest that miRNA let-7b reduces CYP2J2 expression, which may contribute to inhibiting tumor phenotypes. PMID:22761738

  19. An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

    PubMed Central

    Flytzanis, Nicholas C.; Balsamo, Michele; Condeelis, John S.; Oktay, Maja H.; Burge, Christopher B.; Gertler, Frank B.

    2011-01-01

    Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression. PMID:21876675

  20. The kinetochore protein, CENPF, is mutated in human ciliopathy and microcephaly phenotypes

    PubMed Central

    Waters, Aoife M; Asfahani, Rowan; Carroll, Paula; Bicknell, Louise; Lescai, Francesco; Bright, Alison; Chanudet, Estelle; Brooks, Anthony; Christou-Savina, Sonja; Osman, Guled; Walsh, Patrick; Bacchelli, Chiara; Chapgier, Ariane; Vernay, Bertrand; Bader, David M; Deshpande, Charu; O’ Sullivan, Mary; Ocaka, Louise; Stanescu, Horia; Stewart, Helen S; Hildebrandt, Friedhelm; Otto, Edgar; Johnson, Colin A; Szymanska, Katarzyna; Katsanis, Nicholas; Davis, Erica; Kleta, Robert; Hubank, Mike; Doxsey, Stephen; Jackson, Andrew; Stupka, Elia; Winey, Mark; Beales, Philip L

    2015-01-01

    Background Mutations in microtubule-regulating genes are associated with disorders of neuronal migration and microcephaly. Regulation of centriole length has been shown to underlie the pathogenesis of certain ciliopathy phenotypes. Using a next-generation sequencing approach, we identified mutations in a novel centriolar disease gene in a kindred with an embryonic lethal ciliopathy phenotype and in a patient with primary microcephaly. Methods and results Whole exome sequencing data from a non-consanguineous Caucasian kindred exhibiting mid-gestation lethality and ciliopathic malformations revealed two novel non-synonymous variants in CENPF, a microtubule-regulating gene. All four affected fetuses showed segregation for two mutated alleles [IVS5-2A>C, predicted to abolish the consensus splice-acceptor site from exon 6; c.1744G>T, p.E582X]. In a second unrelated patient exhibiting microcephaly, we identified two CENPF mutations [c.1744G>T, p.E582X; c.8692 C>T, p.R2898X] by whole exome sequencing. We found that CENP-F colocalised with Ninein at the subdistal appendages of the mother centriole in mouse inner medullary collecting duct cells. Intraflagellar transport protein-88 (IFT-88) colocalised with CENP-F along the ciliary axonemes of renal epithelial cells in age-matched control human fetuses but did not in truncated cilia of mutant CENPF kidneys. Pairwise co-immunoprecipitation assays of mitotic and serum-starved HEKT293 cells confirmed that IFT88 precipitates with endogenous CENP-F. Conclusions Our data identify CENPF as a new centriolar disease gene implicated in severe human ciliopathy and microcephaly related phenotypes. CENP-F has a novel putative function in ciliogenesis and cortical neurogenesis. PMID:25564561

  1. Shaping the Future for Children with Foetal Alcohol Spectrum Disorders

    ERIC Educational Resources Information Center

    Blackburn, Carolyn; Carpenter, Barry; Egerton, Jo

    2010-01-01

    This article describes work undertaken in connection with an ongoing research project funded by the Training and Development Agency for Schools. It illustrates the educational implications of foetal alcohol spectrum disorders (FASD) and its implications for the educational workforce in seeking to meet the needs of those children who are affected.

  2. Psychiatric outcome of termination of pregnancy for foetal abnormality.

    PubMed

    Iles, S; Gath, D

    1993-05-01

    Termination of pregnancy for foetal abnormality has become frequent with the increasing sophistication of techniques of antenatal diagnosis. The aim of this study was to obtain quantitative and qualitative information about psychiatric morbidity in women after termination of pregnancy for foetal abnormality. Two samples of women were compared. The first consisted of 71 women who had had a termination of pregnancy for foetal abnormality (FA group). The second consisted of 26 women who had experienced so-called missed abortion (MA group). Both groups had lost a pregnancy in the mid-trimester of pregnancy, but the MA group had no element of choice. Standardized psychiatric and social measures were used to assess both groups on three occasions after the termination. In both groups, 4 weeks after the termination psychiatric morbidity was high (four to five times higher than in the general population of women), and social adjustment was impaired. Six months and 12 months after the abortion, levels of psychiatric morbidity were near normal. Semi-structured interviewing was used to obtain information about the experience of grief after mid-trimester termination. For many women, symptoms of grief persisted throughout the year. These symptoms included typical features of grief as well as grief symptoms specific to pregnancy loss. The findings have implications for the counselling of women after termination for foetal abnormality or after missed abortion. PMID:8332657

  3. Phenotypic and genotypic characterization of Vagococcus fluvialis, including strains isolated from human sources.

    PubMed Central

    Teixeira, L M; Carvalho, M G; Merquior, V L; Steigerwalt, A G; Brenner, D J; Facklam, R R

    1997-01-01

    This study presents phenotypic and genotypic data for seven isolates of Vagococcus fluvialis, including four strains recovered from human clinical sources, one strain isolated from an environmental source, and two strains isolated from pigs. On the basis of phenotypic characteristics, most isolates were initially classified as "unidentified enterococci," because they resembled atypical arginine-negative enterococcal species. All seven strains as well as the type strain of V. fluvialis reacted with the AccuProbe Enterococcus genetic probe. The seven isolates had virtually indistinguishable whole-cell protein profiles that were similar to that of the V. fluvialis type strain and distinct from those of Enterococcus and Lactococcus species. DNA-DNA reassociation experiments confirmed that the strains were V. fluvialis. They were 71% or more related to the V. fluvialis type strain under optimum and stringent conditions, with 2.5% or less divergence within related sequences. All strains were susceptible to ampicillin, cefotaxime, trimethoprim-sulfamethoxazole, and vancomycin and were resistant to clindamycin, lomefloxacin, and ofloxacin. Strain-to-strain variation was observed in relation to susceptibilities to 18 other antimicrobial agents. Chromosomal DNA was analyzed by pulsed-field gel electrophoresis (PFGE) after digestion with SmaI. Distinctive PFGE patterns were generated, suggesting the nonclonal nature of V. fluvialis strains. Although the number of strains was small, this report provides molecular characterization of V. fluvialis and the first evidence of a possible connection of this species with human infections. PMID:9350732

  4. Adult human arterial smooth muscle cells in primary culture. Modulation from contractile to synthetic phenotype.

    PubMed

    Thyberg, J; Nilsson, J; Palmberg, L; Sjölund, M

    1985-01-01

    Smooth muscle cells were isolated enzymatically from adult human arteries, grown in primary culture in medium containing 10% whole blood serum, and studied by transmission electron microscopy and [3H]thymidine autoradiography. In the intact arterial wall and directly after isolation, each smooth muscle cell had a nucleus with a wide peripheral zone of condensed chromatin and a cytoplasm dominated by myofilament bundles with associated dense bodies. After 1-2 days of culture, the cells had attached to the substrate and started to spread out. At the same time, a characteristic fine-structural modification took place. It included nuclear enlargement, dispersion of the chromatin and formation of large nucleoli. Moreover, myofilament bundles disappeared and an extensive rough endoplasmic reticulum and a large Golgi complex were organized in the cytoplasm. This morphological transformation of the cells was completed in 3-4 days. It was accompanied by initiation of DNA replication and mitosis. The observations demonstrate that adult human arterial smooth muscle cells, when cultivated in vitro, pass through a phenotypic modulation of the same type as arterial smooth muscle cells from experimental animals. This modulation gives the cells morphological and functional properties resembling those of the modified smooth muscle cells found in fibroproliferative lesions of atherosclerosis. Further studies of the regulation of smooth muscle phenotype and growth may provide important clues for a better understanding of the pathogenesis of atherosclerosis. PMID:3967287

  5. IL-33 induces a hypo-responsive phenotype in human and mouse mast cells

    PubMed Central

    Jung, Mi-Yeon; Smrž, Daniel; Desai, Avanti; Bandara, Geethani; Ito, Tomonobu; Iwaki, Shoko; Kang, Jeong-Han; Andrade, Marcus V.; Hilderbrand, Susana C.; Brown, Jared M.; Beaven, Michael A.; Metcalfe, Dean D.; Gilfillan, Alasdair M.

    2012-01-01

    SUMMARY IL-33 is elevated in afflicted tissues of patients with mast cell-dependent chronic allergic diseases. Based on its acute effects on mouse mast cells (MCs), IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. We now find that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to antigen. This reduction required >72 h exposure to IL-33 for onset and 1–2 wk for reversion following IL-33 removal. This hypo-responsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation including antigen-mediated calcium mobilization and cytoskeletal reorganization; potentially as a consequence of down-regulation of the expression of PLCγ1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to down-regulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease. PMID:23248261

  6. Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

    PubMed Central

    Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

    2015-01-01

    Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. PMID:26182374

  7. Predominance of the metastatic phenotype in hybrids formed by fusion of mouse and human melanoma clones.

    PubMed

    van Golen, K L; Risin, S; Staroselsky, A; Berger, D; Tainsky, M A; Pathak, S; Price, J E

    1996-03-01

    The fusion of mouse and human melanoma cells that were tumorigenic but had different metastatic capabilities resulted in hybrids that were metastatic when injected intravenously or subcutaneously into nude mice, regardless of whether it was the mouse or the human melanoma clone that was metastatic. The H7 hybrid line, formed by fusing murine nonmetastatic K1735 C19 cells with human metastatic A375 C15 cells retained high metastatic potential over more than 50 sub-culture passages, suggesting that the dominant metastatic phenotype in these hybrid cells was stable. Using fluorescent in situ hybridization (FISH), human chromosome 17 was consistently identified as the predominant human chromosome in the majority of H7 cells tested between passages 20 and 60. Western blot analysis showed that the hybrid cells expressed human nm23 protein, indicating that at least one gene on the human chromosome 17 was functional. Immunocytochemistry and immunoprecipitation showed that the metastatic A375 C15 and H7 cells expressed p53 protein, but that the nonmetastatic K1735 C19 melanoma cells did not. Sequencing the human p53 gene in A375 C15N and H7 showed mutations in exon 7. Using a bioassay technique, we showed that K1735 C19 cells can spread from subcutaneous tumors to the lungs of nude mice yet fail to form metastases. With the addition of human chromosome 17 from A375 C15 cells, which carries a mutant p53 gene, the cells readily formed lung metastases. In this melanoma hybrid, a mutant p53 gene appears to confer a survival advantage on cells arrested in the lungs of nude mice and thus contributes to the growth of metastatic cells. PMID:8605733

  8. A phenotypic in vitro model for the main determinants of human whole heart function.

    PubMed

    Stancescu, Maria; Molnar, Peter; McAleer, Christopher W; McLamb, William; Long, Christopher J; Oleaga, Carlota; Prot, Jean-Matthieu; Hickman, James J

    2015-08-01

    This article details the construction and testing of a phenotypic assay system that models in vivo cardiac function in a parallel in vitro environment with human stem cell derived cardiomyocytes. The major determinants of human whole-heart function were experimentally modeled by integrating separate 2D cellular systems with BioMicroelectromechanical Systems (BioMEMS) constructs. The model features a serum-free defined medium to enable both acute and chronic evaluation of drugs and toxins. The integration of data from both systems produced biologically relevant predictions of cardiac function in response to varying concentrations of selected drugs. Sotalol, norepinephrine and verapamil were shown to affect the measured parameters according to their specific mechanism of action, in agreement with clinical data. This system is applicable for cardiac side effect assessment, general toxicology, efficacy studies, and evaluation of in vitro cellular disease models in body-on-a-chip systems. PMID:25978005

  9. A phenotypic in vitro model for the main determinants of human whole heart function

    PubMed Central

    Stancescu, Maria; Molnar, Peter; McAleer, Christopher W.; McLamb, William; Long, Christopher J.; Oleaga, Carlota; Prot, Jean-Matthieu; Hickman, James J.

    2015-01-01

    This article details the construction and testing of a phenotypic assay system that models in vivo cardiac function in a parallel in vitro environment with human stem cell derived cardiomyocytes. The major determinants of human whole-heart function were experimentally modeled by integrating separate 2D cellular systems with BioMicroelectromechanical Systems (BioMEMS) constructs. The model featured a serum-free defined medium to enable both acute and chronic evaluation of drugs and toxins. The integration of data from both systems produced biologically relevant predictions of cardiac function in response to varying concentrations of selected drugs. Sotalol, norepinephrine and verapamil were shown to affect the measured parameters according to their specific mechanism of action, in agreement with clinical data. This system is applicable for cardiac side effect assessment, general toxicology, efficacy studies, and evaluation of in vitro cellular disease models in body-on-a-chip systems. PMID:25978005

  10. Accelerated cellular senescence phenotype of GAPDH-depleted human lung carcinoma cells

    SciTech Connect

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Krynetskiy, Evgeny

    2011-07-29

    Highlights: {yields} We examined the effect of glyceraldehyde 3-phosphate (GAPDH) depletion on proliferation of human carcinoma A549 cells. {yields} GAPDH depletion induces accelerated senescence in tumor cells via AMPK network, in the absence of DNA damage. {yields} Metabolic and genetic rescue experiments indicate that GAPDH has regulatory functions linking energy metabolism and cell cycle. {yields} Induction of senescence in LKB1-deficient lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation. -- Abstract: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a pivotal glycolytic enzyme, and a signaling molecule which acts at the interface between stress factors and the cellular apoptotic machinery. Earlier, we found that knockdown of GAPDH in human carcinoma cell lines resulted in cell proliferation arrest and chemoresistance to S phase-specific cytotoxic agents. To elucidate the mechanism by which GAPDH depletion arrests cell proliferation, we examined the effect of GAPDH knockdown on human carcinoma cells A549. Our results show that GAPDH-depleted cells establish senescence phenotype, as revealed by proliferation arrest, changes in morphology, SA-{beta}-galactosidase staining, and more than 2-fold up-regulation of senescence-associated genes DEC1 and GLB1. Accelerated senescence following GAPDH depletion results from compromised glycolysis and energy crisis leading to the sustained AMPK activation via phosphorylation of {alpha} subunit at Thr172. Our findings demonstrate that GAPDH depletion switches human tumor cells to senescent phenotype via AMPK network, in the absence of DNA damage. Rescue experiments using metabolic and genetic models confirmed that GAPDH has important regulatory functions linking the energy metabolism and the cell cycle networks. Induction of senescence in LKB1-deficient non-small cell lung cancer cells via GAPDH depletion suggests a novel strategy to control tumor cell proliferation.

  11. Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

    PubMed Central

    Latif, Najma; Quillon, Alfred; Sarathchandra, Padmini; McCormack, Ann; Lozanoski, Alec; Yacoub, Magdi H.; Chester, Adrian H.

    2015-01-01

    Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the

  12. Successful maternal-foetal outcome using nitric oxide and sildenafil in pulmonary hypertension with atrial septal defect and HIV infection.

    PubMed

    Ng, W P; Yip, W L

    2012-01-01

    Pulmonary hypertension associated with pregnancy carries a poor prognosis. We describe successful maternal-foetal outcome for a 30-year-old woman who was found to have severe pulmonary hypertension, human immunodeficiency virus (HIV) and an atrial septal defect. Prior to delivery, she was managed with subcutaneous enoxaparine, sildenafil, nitric oxide, careful maintenance of a euvolemic status and antiretroviral therapy. She was planned for an elective Caesarean section to reduce the risk of maternal-foetal HIV transmission, but went into labour in the coronary care unit. During delivery, antibiotic prophylaxis was given, although there was insufficient time for intravenous zidovudine. Peripartum, the patient was continued on nitric oxide and subcutaneous enoxaparine. She was eventually weaned off the nitric oxide and recovered well. PMID:22252195

  13. Location and phenotype of human adult keratinocyte stem cells of the skin.

    PubMed

    Webb, Angela; Li, Amy; Kaur, Pritinder

    2004-10-01

    The location and identity of interfollicular epidermal stem cells of adult human skin remain undefined. Based on our previous work in both adult murine and neonatal human foreskin, we demonstrate that cell surface levels of the alpha6 integrin and the transferrin receptor (CD71) are valid markers for resolving a putative stem cell, transit amplifying and differentiating compartment in adult human skin by flow cytometry. Specifically, epidermal cells expressing high levels of alpha6 integrin and low levels of the transferrin receptor CD71 (phenotype alpha6 (bri)CD71(dim)) exhibit several stem cell characteristics, comprising a minor population (2%-5%) of the K14(bri) fraction, enriched for quiescent and small blast-like cells with high clonogenic capacity, lacking the differentiation marker K10. Conversely, the majority of K14(bri) K10(neg) epidermal cells express high levels of CD71 (phenotype alpha6 (bri)CD71(bri)), and represent the actively cycling fraction of keratinocytes displaying greater cell size due to an increase in cytoplasmic area, consistent with their being transient amplifying cells. The alpha6 (bri)CD71(bri) population exhibited intermediate clonogenic capacity. A third population of K14(dim) but K10 positive epidermal cells could be identified by their low levels of alpha6 integrin expression (i.e. alpha6 (dim) cells), representing the differentiation compartment; predictably, this subpopulation exhibited poor clonogenic efficiency. Flow cytometric analysis for the hair follicle bulge region (stem cell) marker K15 revealed preferential expression of this keratin in alpha6 (bri) cells (i.e., both stem and transient amplifying fractions), but not the alpha6 (dim) population. Given that K15 positive cells could only be detected in the deep rete ridges of adult skin in situ, we conclude that stem and transient amplifying cells reside in this location, while differentiating (K15 negative) cells are found in the shallow rete ridges. PMID:15606498

  14. Phenotypic evolution of human craniofacial morphology after admixture: a geometric morphometrics approach.

    PubMed

    Martínez-Abadías, Neus; González-José, Rolando; González-Martín, Antonio; Van der Molen, Silvina; Talavera, Arturo; Hernández, Patricia; Hernández, Miquel

    2006-03-01

    An evolutionary, diachronic approach to the phenotypic craniofacial pattern arisen in a human population after high levels of admixture and gene flow was achieved by means of geometric morphometrics. Admixture has long been studied after molecular data. Nevertheless, few efforts have been made to explain the morphological outcome in human craniofacial samples. The Spanish-Amerindian contact can be considered a good scenario for such an analysis. Here we present a comparative analysis of craniofacial shape changes observed between two putative ancestor groups, Spanish and precontact Aztecs, and two diachronic admixed groups, corresponding to early and late colonial periods from the Mexico's Central Valley. Quantitative shape comparisons of Amerindian, Spanish, and admixed groups were used to test the expectations of quantitative genetics for admixture events. In its simplest form, this prediction states that an admixed group will present phenotypic values falling between those of both parental groups. Results show that, in general terms, although the human skull is a complex, integrated structure, the craniofacial morphology observed fits the theoretical expectations of quantitative genetics. Thus, it is predictive of population structure and history. In fact, results obtained after the craniofacial analysis are in accordance with previous molecular and historical interpretations, providing evidence that admixture is a main microevolutionary agent influencing modern Mexican gene pool. However, expectations are not straightforward when moderate shape changes are considered. Deviations detected at localized structures, such as the upper and lower face, highlight the evolution of a craniofacial pattern exclusively inherent to the admixed groups, indicating that quantitative characters might respond to admixture in a complicated, nondirectional way. PMID:16323202

  15. Transformation of human osteoblast cells to the tumorigenic phenotype by depleted uranium-uranyl chloride.

    PubMed Central

    Miller, A C; Blakely, W F; Livengood, D; Whittaker, T; Xu, J; Ejnik, J W; Hamilton, M M; Parlette, E; John, T S; Gerstenberg, H M; Hsu, H

    1998-01-01

    Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Although the health effects of occupational uranium exposure are well known, limited data exist regarding the long-term health effects of internalized DU in humans. We established an in vitro cellular model to study DU exposure. Microdosimetric assessment, determined using a Monte Carlo computer simulation based on measured intracellular and extracellular uranium levels, showed that few (0.0014%) cell nuclei were hit by alpha particles. We report the ability of DU-uranyl chloride to transform immortalized human osteoblastic cells (HOS) to the tumorigenic phenotype. DU-uranyl chloride-transformants are characterized by anchorage-independent growth, tumor formation in nude mice, expression of high levels of the k-ras oncogene, reduced production of the Rb tumor-suppressor protein, and elevated levels of sister chromatid exchanges per cell. DU-uranyl chloride treatment resulted in a 9.6 (+/- 2.8)-fold increase in transformation frequency compared to untreated cells. In comparison, nickel sulfate resulted in a 7.1 (+/- 2.1)-fold increase in transformation frequency. This is the first report showing that a DU compound caused human cell transformation to the neoplastic phenotype. Although additional studies are needed to determine if protracted DU exposure produces tumors in vivo, the implication from these in vitro results is that the risk of cancer induction from internalized DU exposure may be comparable to other biologically reactive and carcinogenic heavy-metal compounds (e.g., nickel). Images Figure 1 Figure 2 Figure 3 PMID:9681973

  16. Classification of Human Chromosome 21 Gene-Expression Variations in Down Syndrome: Impact on Disease Phenotypes

    PubMed Central

    Aït Yahya-Graison, E. ; Aubert, J. ; Dauphinot, L. ; Rivals, I. ; Prieur, M. ; Golfier, G. ; Rossier, J. ; Personnaz, L. ; Créau, N. ; Bléhaut, H. ; Robin, S. ; Delabar, J. M. ; Potier, M.-C. 

    2007-01-01

    Down syndrome caused by chromosome 21 trisomy is the most common genetic cause of mental retardation in humans. Disruption of the phenotype is thought to be the result of gene-dosage imbalance. Variations in chromosome 21 gene expression in Down syndrome were analyzed in lymphoblastoid cells derived from patients and control individuals. Of the 359 genes and predictions displayed on a specifically designed high-content chromosome 21 microarray, one-third were expressed in lymphoblastoid cells. We performed a mixed-model analysis of variance to find genes that are differentially expressed in Down syndrome independent of sex and interindividual variations. In addition, we identified genes with variations between Down syndrome and control samples that were significantly different from the gene-dosage effect (1.5). Microarray data were validated by quantitative polymerase chain reaction. We found that 29% of the expressed chromosome 21 transcripts are overexpressed in Down syndrome and correspond to either genes or open reading frames. Among these, 22% are increased proportional to the gene-dosage effect, and 7% are amplified. The other 71% of expressed sequences are either compensated (56%, with a large proportion of predicted genes and antisense transcripts) or highly variable among individuals (15%). Thus, most of the chromosome 21 transcripts are compensated for the gene-dosage effect. Overexpressed genes are likely to be involved in the Down syndrome phenotype, in contrast to the compensated genes. Highly variable genes could account for phenotypic variations observed in patients. Finally, we show that alternative transcripts belonging to the same gene are similarly regulated in Down syndrome but sense and antisense transcripts are not. PMID:17701894

  17. Expression of mutant cartilage oligomeric matrix protein in human chondrocytes induces the pseudoachondroplasia phenotype.

    PubMed

    Merritt, Thomas M; Alcorn, Joseph L; Haynes, Richard; Hecht, Jacqueline T

    2006-04-01

    Over 70 mutations in the cartilage oligomeric matrix protein (COMP), a large extracellular pentameric glycoprotein synthesized by chondrocytes, have been identified as causing two skeletal dysplasias: multiple epiphyseal dysplasia (MED/EDM1), and a dwarfing condition, pseudoachondroplasia (PSACH). These mutations induce misfolding of intracellular COMP, resulting in retention of the protein in the rough endoplasmic reticulum (rER) of chondrocytes. This accumulation of COMP in the rER creates the phenotypic enlarged rER cisternae in the cells, which is believed to compromise chondrocyte function and eventually cause cell death. To study the molecular mechanisms involved with the disease, we sought to develop an in vitro model that recapitulates the PSACH phenotype. Normal human chondrocytes were transfected with wildtype (wt-) COMP or with mutant COMP (D469del; mt-) recombinant adenoviruses and grown in a nonattachment redifferentiating culture system that provides an environment allowing formation of a differentiated chondrocyte nodule. Visualization of normal cells expressing COMP suggested the hallmarks of the PSACH phenotype. Mutant COMP expressed in normal cells was retained in enlarged rER cisternae, which also retained IX collagen (COL9) and matrilin-3 (MATN3). Although these proteins were secreted normally into the ECM of the wt-COMP nodules, reduced secretion of these proteins was observed in nodules composed of cells transfected with mt-COMP. The findings complement those found in chondrocytes from PSACH patient growth plates. This new model system allows for production of PSACH chondrocyte pathology in normal costochondral chondrocytes and can be used for future mechanistic and potential gene therapy studies. PMID:16514635

  18. A Computational Protein Phenotype Prediction Approach to Analyze the Deleterious Mutations of Human MED12 Gene.

    PubMed

    Banaganapalli, Babajan; Mohammed, Kaleemuddin; Khan, Imran Ali; Al-Aama, Jumana Y; Elango, Ramu; Shaik, Noor Ahmad

    2016-09-01

    Genetic mutations in MED12, a subunit of Mediator complex are seen in a broad spectrum of human diseases. However, the underlying basis of how these pathogenic mutations elicit protein phenotype changes in terms of 3D structure, stability and protein binding sites remains unknown. Therefore, we aimed to investigate the structural and functional impacts of MED12 mutations, using computational methods as an alternate to traditional in vivo and in vitro approaches. The MED12 gene mutations details and their corresponding clinical associations were collected from different databases and by text-mining. Initially, diverse computational approaches were applied to categorize the different classes of mutations based on their deleterious impact to MED12. Then, protein structures for wild and mutant types built by integrative modeling were analyzed for structural divergence, solvent accessibility, stability, and functional interaction deformities. Finally, this study was able to identify that genetic mutations mapped to exon-2 region, highly conserved LCEWAV and Catenin domains induce biochemically severe amino acid changes which alters the protein phenotype as well as the stability of MED12-CYCC interactions. To better understand the deleterious nature of FS-IDs and Indels, this study asserts the utility of computational screening based on their propensity towards non-sense mediated decay. Current study findings may help to narrow down the number of MED12 mutations to be screened for mediator complex dysfunction associated genetic diseases. This study supports computational methods as a primary filter to verify the plausible impact of pathogenic mutations based on the perspective of evolution, expression and phenotype of proteins. J. Cell. Biochem. 117: 2023-2035, 2016. © 2016 Wiley Periodicals, Inc. PMID:26813965

  19. Phenotypic and Genomic Analysis of Hypervirulent Human-associated Bordetella bronchiseptica

    PubMed Central

    2012-01-01

    Background B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics. Results Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates. Conclusion Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage. PMID:22863321

  20. A global map for dissecting phenotypic variants in human lincRNAs.

    PubMed

    Ning, Shangwei; Wang, Peng; Ye, Jingrun; Li, Xiang; Li, Ronghong; Zhao, Zuxianglan; Huo, Xiao; Wang, Li; Li, Feng; Li, Xia

    2013-10-01

    Large intergenic noncoding RNAs (lincRNAs) are emerging as key factors of multiple cellular processes. Cumulative evidence has linked lincRNA polymorphisms to diverse diseases. However, the global properties of lincRNA polymorphisms and their implications for human disease remain largely unknown. Here we performed a systematic analysis of naturally occurring variants in human lincRNAs, with a particular focus on lincRNA polymorphism as novel risk factor of disease etiology. We found that lincRNAs exhibited a relatively low level of polymorphisms, and low single-nucleotide polymorphism (SNP) density lincRNAs might have a broad range of functions. We also found that some polymorphisms in evolutionarily conserved regions of lincRNAs had significant effects on predicted RNA secondary structures, indicating their potential contribution to diseases. We mapped currently available phenotype-associated SNPs to lincRNAs and found that lincRNAs were associated with a wide range of human diseases. Some lincRNAs could be responsible for particular diseases. Our results provided not only a global perspective on genetic variants in human lincRNAs but also novel insights into the function and etiology of lincRNA. All the data in this study can be accessed and retrieved freely via a web server at http://bioinfo.hrbmu.edu.cn/lincPoly. PMID:23463026

  1. Human Cerberus Prevents Nodal-Receptor Binding, Inhibits Nodal Signaling, and Suppresses Nodal-Mediated Phenotypes

    PubMed Central

    Aykul, Senem; Ni, Wendi; Mutatu, Washington; Martinez-Hackert, Erik

    2015-01-01

    The Transforming Growth Factor-ß (TGFß) family ligand Nodal is an essential embryonic morphogen that is associated with progression of breast and other cancers. It has therefore been suggested that Nodal inhibitors could be used to treat breast cancers where Nodal plays a defined role. As secreted antagonists, such as Cerberus, tightly regulate Nodal signaling during embryonic development, we undertook to produce human Cerberus, characterize its biochemical activities, and determine its effect on human breast cancer cells. Using quantitative methods, we investigated the mechanism of Nodal signaling, we evaluated binding of human Cerberus to Nodal and other TGFß family ligands, and we characterized the mechanism of Nodal inhibition by Cerberus. Using cancer cell assays, we examined the ability of Cerberus to suppress aggressive breast cancer cell phenotypes. We found that human Cerberus binds Nodal with high affinity and specificity, blocks binding of Nodal to its signaling partners, and inhibits Nodal signaling. Moreover, we showed that Cerberus profoundly suppresses migration, invasion, and colony forming ability of Nodal expressing and Nodal supplemented breast cancer cells. Taken together, our studies provide mechanistic insights into Nodal signaling and Nodal inhibition with Cerberus and highlight the potential value of Cerberus as anti-Nodal therapeutic. PMID:25603319

  2. Phenotypic and Functional Characterization of Long-Term Cryopreserved Human Adipose-derived Stem Cells

    PubMed Central

    Yong, Kar Wey; Pingguan-Murphy, Belinda; Xu, Feng; Abas, Wan Abu Bakar Wan; Choi, Jane Ru; Omar, Siti Zawiah; Azmi, Mat Adenan Noor; Chua, Kien Hui; Safwani, Wan Kamarul Zaman Wan

    2015-01-01

    Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications. PMID:25872464

  3. Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype.

    PubMed

    Brennan, Kiva; McSharry, Brian P; Keating, Sinéad; Petrasca, Andreea; O'Reilly, Vincent P; Keane, Joseph; Doherty, Derek G; Gardiner, Clair M

    2016-10-01

    NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production. Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells. ULBP2 expression was however linked to expression of mature CD57(+) NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated "mature" NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells. PMID:27349945

  4. Induction of predominant tenogenic phenotype in human dermal fibroblasts via synergistic effect of TGF-β and elongated cell shape.

    PubMed

    Wang, Wenbo; Li, Jie; Wang, Keyun; Zhang, Zhiyong; Zhang, Wenjie; Zhou, Guangdong; Cao, Yilin; Ye, Mingliang; Zou, Hanfa; Liu, Wei

    2016-03-01

    Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling. PMID:26632599

  5. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    PubMed

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  6. Stress Signaling from Human Mammary Epithelial Cells Contributes to Phenotypes of Mammographic Density

    PubMed Central

    Patten, Kelley; Chang, Hang; Zhao, Jianxin; Fontenay, Gerald V.; Kerlikowske, Karla; Parvin, Bahram; Tlsty, Thea D.

    2014-01-01

    Telomere malfunction and other types of DNA damage induce an activin A-dependent stress response in mortal non-tumorigenic human mammary epithelial cells that subsequently induces desmoplastic-like phenotypes in neighboring fibroblasts. Some characteristics of this fibroblast/stromal response, such as reduced adipocytes and increased extracellular matrix content, are observed not only in tumor tissues but also in disease-free breast tissues at high risk for developing cancer, especially high mammographic density tissues. We found that these phenotypes are induced by repression of the fatty acid translocase CD36, which is seen in desmoplastic and disease-free high mammographic density tissues. In this study, we show that epithelial cells from high mammographic density tissues have more DNA damage signaling, shorter telomeres, increased activin A secretion and an altered DNA damage response compared to epithelial cells from low mammographic density tissues. Strikingly, both telomere malfunction and activin A expression in epithelial cells can repress CD36 expression in adjacent fibroblasts. These results provide new insights into how high mammographic density arises and why it is associated with breast cancer risk, with implications for the definition of novel invention targets (e.g. activin A, CD36) to prevent breast cancer. PMID:25172842

  7. Human lymph-node CD8+ T cells display an altered phenotype during systemic autoimmunity

    PubMed Central

    Ramwadhdoebe, Tamara H; Hähnlein, Janine; van Kuijk, Bo J; Choi, Ivy Y; van Boven, Leonard J; Gerlag, Danielle M; Tak, Paul P; van Baarsen, Lisa G

    2016-01-01

    Although many studies are focused on auto-reactive CD4+ T cells, the precise role of CD8+ T cells in autoimmunity is poorly understood. The objective of this study is to provide more insight into the phenotype and function CD8+ T cells during the development of autoimmune disease by studying CD8+ T cells in human lymph-node biopsies and peripheral blood obtained during the earliest phases of rheumatoid arthritis (RA). Here, we show that lymphoid pro-inflammatory CD8+ T cells exhibit a less-responsive phenotype already during the earliest phases of autoimmunity compared with healthy individuals. We found an increase in CD8+ memory T cells in lymphoid tissue during the earliest phases of autoimmunity, even before clinical onset of RA, accompanied by an increased frequency of non-circulating or recently activated (CD69+) CD8+ T cells in lymphoid tissue and peripheral blood. Importantly, lymphoid pro-inflammatory CD8+IL-17A+ T cells displayed a decreased capacity of cytokine production, which was related to disease activity in early RA patients. In addition, a decreased frequency of regulatory CD8+IL-10+ T cells in peripheral blood was also related to disease activity in early RA patients. Our results suggest that different CD8+ T-cell subsets are affected already during the earliest phases of systemic autoimmunity. PMID:27195110

  8. Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

    PubMed Central

    Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

    2010-01-01

    Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

  9. Lactate Activates HIF-1 in Oxidative but Not in Warburg-Phenotype Human Tumor Cells

    PubMed Central

    De Saedeleer, Christophe J.; Copetti, Tamara; Porporato, Paolo E.; Verrax, Julien

    2012-01-01

    Cancer can be envisioned as a metabolic disease driven by pressure selection and intercellular cooperativeness. Together with anaerobic glycolysis, the Warburg effect, formally corresponding to uncoupling glycolysis from oxidative phosphorylation, directly participates in cancer aggressiveness, supporting both tumor progression and dissemination. The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key contributor to glycolysis. It stimulates the expression of glycolytic transporters and enzymes supporting high rate of glycolysis. In this study, we addressed the reverse possibility of a metabolic control of HIF-1 in tumor cells. We report that lactate, the end-product of glycolysis, inhibits prolylhydroxylase 2 activity and activates HIF-1 in normoxic oxidative tumor cells but not in Warburg-phenotype tumor cells which also expressed lower basal levels of HIF-1α. These data were confirmed using genotypically matched oxidative and mitochondria-depleted glycolytic tumor cells as well as several different wild-type human tumor cell lines of either metabolic phenotype. Lactate activates HIF-1 and triggers tumor angiogenesis and tumor growth in vivo, an activity that we found to be under the specific upstream control of the lactate transporter monocarboxylate transporter 1 (MCT1) expressed in tumor cells. Because MCT1 also gates lactate-fueled tumor cell respiration and mediates pro-angiogenic lactate signaling in endothelial cells, MCT1 inhibition is confirmed as an attractive anticancer strategy in which a single drug may target multiple tumor-promoting pathways. PMID:23082126

  10. Morphologic and phenotypic changes of human neuroblastoma cells in culture induced by cytosine arabinoside

    SciTech Connect

    Ponzoni, M.; Lanciotti, M.; Melodia, A.; Casalaro, A.; Cornaglia-Ferraris, P. )

    1989-03-01

    The effects of cytosine-arabinoside (ARA-C) on the growth and phenotypic expression of a new human neuroblastoma (NB) cell line (GI-ME-N) have been extensively tested. Low doses of ARA-C allowing more than 90% cell viability induce morphological differentiation and growth inhibition. Differentiated cells were larger and flattened with elongated dendritic processes; such cells appeared within 48 hours after a dose of ARA-C as low as 0.1 {mu}g/ml. The new morphological aspect reached the maximum expression after 5-6 days of culture being independent from the addition of extra drug to the culture. A decrease in ({sup 3}H)thymidine incorporation was also observed within 24 hours and the cell growth was completely inhibited on the sixth day. Moreover, ARA-C strongly inhibited anchorage-independent growth in soft agar assay. Membrane immunofluorescence showed several dramatic changes in NB-specific antigen expression after 5 days of treatment with ARA-C. At the same time ARA-C also modulated cytoskeletal proteins and slightly increased catecholamine expression. These findings suggest that noncytotoxic doses of ARA-C do promote the differentiation of GI-ME-N neuroblastoma cells associated with reduced expression of the malignant phenotype.

  11. Evaluation of Proposed In Vivo Probe Substrates and Inhibitors for Phenotyping Transporter Activity in Humans.

    PubMed

    Momper, Jeremiah D; Tsunoda, Shirley M; Ma, Joseph D

    2016-07-01

    Drug transporters are present in various tissues and have a significant role in drug absorption, distribution, and elimination. The International Transporter Consortium has identified 7 transporters of increasing importance from evidence of clinically significant transporter-mediated drug-drug interactions. The transporters are P-glycoprotein, breast cancer resistance protein, organic anion transporting polypeptide (OATP) 1B1, OATP1B3, organic cation transporter 2, organic anion transporters (OAT) 1, and OAT3. Decision trees were created based on in vitro experiments to determine whether an in vivo transporter-mediated drug-drug interaction study is needed. Phenotyping is a methodology that evaluates real-time in vivo transporter activity, whereby changes in a probe substrate or probe inhibitor reflect alternations in the activity of the specified transporter. In vivo probe substrates and/or probe inhibitors have been proposed for each aforementioned transporter. In vitro findings and animal models provide the strongest evidence regarding probe specificity. However, such findings have not conclusively correlated with human phenotyping studies. Furthermore, the extent of contribution from multiple transporters in probe disposition complicates the ability to discern if study findings are the result of a specific transporter and thus provide a recommendation for a preferred probe for a drug transporter. PMID:27385182

  12. Human haptoglobin phenotypes and concentration determination by nanogold-enhanced electrochemical impedance spectroscopy

    NASA Astrophysics Data System (ADS)

    Cheng, Tsai-Mu; Lee, Tzu-Cheng; Tseng, Shin-Hua; Chu, Hsueh-Liang; Pan, Ju-Pin; Chang, Chia-Ching

    2011-06-01

    Haptoglobin (Hp) is an acute phase protein that binds free hemoglobin (Hb), preventing Hb-induced oxidative damage in the vascular system. There are three phenotypes in human Hp, whose heterogeneous polymorphic structures and varying concentrations in plasma have been attributed to the cause of diseases and outcome of clinical treatments. Different phenotypes of Hp may be composed of the same subunits but different copy numbers, rendering their determination difficult by a single procedure. In this study, we have developed a simple, fast, reliable and sensitive method, using label-free nanogold-modified bioprobes coupled with self-development electrochemical impedance spectroscopy (EIS). By this method, probe surface charge transfer resistance is detected. The relative charge transfer resistance ratios for Hp 1-1, Hp 2-1 and Hp 2-2 were characterized. We were able to determine protein size difference within 3 nm, and the linear region of the calibration curve for Hp levels in the range of 90 pg ml - 1 and 90 µg ml - 1 (~1 fM to 1 pM). We surmise that similar approaches can be used to investigate protein polymorphism and altered protein-protein interaction associated with diseases.

  13. G1 Domain of Versican Regulates Hyaluronan Organization and the Phenotype of Cultured Human Dermal Fibroblasts.

    PubMed

    Merrilees, Mervyn J; Zuo, Ning; Evanko, Stephen P; Day, Anthony J; Wight, Thomas N

    2016-06-01

    Variants of versican have wide-ranging effects on cell and tissue phenotype, impacting proliferation, adhesion, pericellular matrix composition, and elastogenesis. The G1 domain of versican, which contains two Link modules that bind to hyaluronan (HA), may be central to these effects. Recombinant human G1 (rhG1) with an N-terminal 8 amino acid histidine (His) tag, produced in Nicotiana benthamiana, was applied to cultures of dermal fibroblasts, and effects on proliferation and pericellular HA organization determined. rhG1 located to individual strands of cell surface HA which aggregated into structures resembling HA cables. On both individual and aggregated strands, the spacing of attached rhG1 was similar (~120 nm), suggesting interaction between rhG1 molecules. Endogenous V0/V1, present on HA between attached rhG1, did not prevent cable formation, while treatment with V0/V1 alone, which also bound to HA, did not induce cables. A single treatment with rhG1 suppressed cell proliferation for an extended period. Treating cells for 4 weeks with rhG1 resulted in condensed layers of elongated, differentiated α actin-positive fibroblasts, with rhG1 localized to cell surfaces, and a compact extracellular matrix including both collagen and elastin. These results demonstrate that the G1 domain of versican can regulate the organization of pericellular HA and affect phenotype. PMID:27126822

  14. The complete local genotype–phenotype landscape for the alternative splicing of a human exon

    PubMed Central

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype–phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function—the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  15. The complete local genotype-phenotype landscape for the alternative splicing of a human exon.

    PubMed

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype-phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function-the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  16. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease.

    PubMed

    Varshney, Gaurav Kumar; Burgess, Shawn Michael

    2014-03-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  17. Cystic fibrosis mice carrying the missense mutation G551D replicate human genotype-phenotype correlations.

    PubMed Central

    Delaney, S J; Alton, E W; Smith, S N; Lunn, D P; Farley, R; Lovelock, P K; Thomson, S A; Hume, D A; Lamb, D; Porteous, D J; Dorin, J R; Wainwright, B J

    1996-01-01

    We have generated a mouse carrying the human G551D mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a one-step gene targeting procedure. These mutant mice show cystic fibrosis pathology but have a reduced risk of fatal intestinal blockage compared with 'null' mutants, in keeping with the reduced incidence of meconium ileus in G551D patients. The G551D mutant mice show greatly reduced CFTR-related chloride transport, displaying activity intermediate between that of cftr(mlUNC) replacement ('null') and cftr(mlHGU) insertional (residual activity) mutants and equivalent to approximately 4% of wild-type CFTR activity. The long-term survival of these animals should provide an excellent model with which to study cystic fibrosis, and they illustrate the value of mouse models carrying relevant mutations for examining genotype-phenotype correlations. Images PMID:8605891

  18. MicroRNAs Induce Epigenetic Reprogramming and Suppress Malignant Phenotypes of Human Colon Cancer Cells.

    PubMed

    Ogawa, Hisataka; Wu, Xin; Kawamoto, Koichi; Nishida, Naohiro; Konno, Masamitsu; Koseki, Jun; Matsui, Hidetoshi; Noguchi, Kozou; Gotoh, Noriko; Yamamoto, Tsuyoshi; Miyata, Kanjiro; Nishiyama, Nobuhiro; Nagano, Hiroaki; Yamamoto, Hirofumi; Obika, Satoshi; Kataoka, Kazunori; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2015-01-01

    Although cancer is a genetic disease, epigenetic alterations are involved in its initiation and progression. Previous studies have shown that reprogramming of colon cancer cells using Oct3/4, Sox2, Klf4, and cMyc reduces cancer malignancy. Therefore, cancer reprogramming may be a useful treatment for chemo- or radiotherapy-resistant cancer cells. It was also reported that the introduction of endogenous small-sized, non-coding ribonucleotides such as microRNA (miR) 302s and miR-369-3p or -5p resulted in the induction of cellular reprogramming. miRs are smaller than the genes of transcription factors, making them possibly suitable for use in clinical strategies. Therefore, we reprogrammed colon cancer cells using miR-302s and miR-369-3p or -5p. This resulted in inhibition of cell proliferation and invasion and the stimulation of the mesenchymal-to-epithelial transition phenotype in colon cancer cells. Importantly, the introduction of the ribonucleotides resulted in epigenetic reprogramming of DNA demethylation and histone modification events. Furthermore, in vivo administration of the ribonucleotides in mice elicited the induction of cancer cell apoptosis, which involves the mitochondrial Bcl2 protein family. The present study shows that the introduction of miR-302s and miR-369s could induce cellular reprogramming and modulate malignant phenotypes of human colorectal cancer, suggesting that the appropriate delivery of functional small-sized ribonucleotides may open a new avenue for therapy against human malignant tumors. PMID:25970424

  19. Differential modulation of human (Caco-2) colon cancer cell line phenotype by short chain fatty acids.

    PubMed

    Basson, M D; Emenaker, N J; Hong, F

    1998-04-01

    Fermentation of dietary fiber within the colonic lumen yields short chain fatty acids (SCFA) such as butyrate, which may modulate colonic mucosal biology and inhibit the development of a malignant phenotype. However, different fibers yield varying proportions of various SCFA. We studied the effects of the three most common SCFA, acetate, butyrate, and propionate, on the proliferation, adhesion, and motility of the human intestinal Caco-2 cell line, as well as the effects of these SCFA on alkaline phosphatase and dipeptidyl dipeptidase specific activity (common laboratory markers of differentiation). In addition, we examined the modulation of c-myc protein and the tyrosine phosphorylation of cellular proteins by these SCFA in order to determine whether the variations in the potency of these three SCFA for phenotypic change extended to variations in effects on intracellular signaling and protooncogene expression. All three SCFA tended to slow proliferation, promote brush border enzyme activity, and inhibit both adhesion to and motility across a type I collagen matrix substrate. However, we observed substantial differences in the potency of these three SCFA with regard to these effects. In particular, butyrate was uniformly more potent than an equimolar concentration of acetate whereas equimolar propionate achieved comparable effects with regard to proliferation and brush border enzyme activity but was intermediate between butyrate and acetate with regard to modulation of cell-matrix interactions. Similarly, the SCFA downregulated c-myc protein levels and modulated the phosphorylation of several intracellular tyrosine phosphoproteins, but the effects of the three SCFA varied substantially for these parameters. These results suggest that the common short chain fatty acids are not equipotent in their effects on human Caco-2 colon cancer cell biology. Such differences in potency could contribute to the observed differences in effects of different dietary fibers in vivo

  20. Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

    PubMed Central

    Gingras, Jacinthe; Smith, Sarah; Matson, David J.; Johnson, Danielle; Nye, Kim; Couture, Lauren; Feric, Elma; Yin, Ruoyuan; Moyer, Bryan D.; Peterson, Matthew L.; Rottman, James B.; Beiler, Rudolph J.; Malmberg, Annika B.; McDonough, Stefan I.

    2014-01-01

    Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain. PMID:25188265

  1. Morphological, genetic and phenotypic comparison between human articular chondrocytes and cultured chondrocytes.

    PubMed

    Mata-Miranda, Mónica Maribel; Martinez-Martinez, Claudia María; Noriega-Gonzalez, Jesús Emmanuel; Paredes-Gonzalez, Luis Enrique; Vázquez-Zapién, Gustavo Jesús

    2016-08-01

    Articular cartilage is an avascular and aneural tissue with limited capacity for regeneration. On large articular lesions, it is recommended to use regenerative medicine strategies, like autologous chondrocyte implantation. There is a concern about morphological changes that chondrocytes suffer once they have been isolated and cultured. Due to the fact that there is little evidence that compares articular cartilage chondrocytes with cultured chondrocytes, in this research we proposed to obtain chondrocytes from human articular cartilage, compare them with themselves once they have been cultured and characterize them through genetic, phenotypic and morphological analysis. Knee articular cartilage samples of 10 mm were obtained, and each sample was divided into two fragments; a portion was used to determine gene expression, and from the other portion, chondrocytes were obtained by enzymatic disaggregation, in order to be cultured and expanded in vitro. Subsequently, morphological, genetic and phenotypic characteristics were compared between in situ (articular cartilage) and cultured chondrocytes. Obtained cultured chondrocytes were rounded in shape, possessing a large nucleus with condensed chromatin and a clear cytoplasm; histological appearance was quite similar to typical chondrocyte. The expression levels of COL2A1 and COL10A1 genes were higher in cultured chondrocytes than in situ chondrocytes; moreover, the expression of COL1A1 was almost undetectable on cultured chondrocytes; likewise, COL2 and SOX9 proteins were detected by immunofluorescence. We concluded that chondrocytes derived from adult human cartilage cultured for 21 days do not tend to dedifferentiate, maintaining their capacity to produce matrix and also retaining their synthesis capacity and morphology. PMID:27094849

  2. Isolation of human dermis derived mesenchymal stem cells using explants culture method: expansion and phenotypical characterization.

    PubMed

    Park, Jeong-Ran; Kim, Eunjeong; Yang, Jungwon; Lee, Hanbyeol; Hong, Seok-Ho; Woo, Heung-Myong; Park, Sung-Min; Na, Sunghun; Yang, Se-Ran

    2015-06-01

    Recent studies have reported that stem cells can be isolated from a wide range of tissues including bone marrow, fatty tissue, adipose tissue and placenta. Moreover, several studies also suggest that skin dermis could serve as a source of stem cells, but are of unclear phenotype. Therefore, we isolated and investigated to determine the potential of stem cell within human skin dermis. We isolated cells from human dermis, termed here as human dermis-derived mesenchymal stem cells (hDMSCs) which is able to be isolated by using explants culture method. Our method has an advantage over the enzymatic method as it is easier, less expensive and less cell damage. hDMSCs were maintained in basal culture media and proliferation potential was measured. hDMSCs were highly proliferative and successfully expanded with no additional growth factor. In addition, hDMSCs revealed normal karyotype and expressed high levels of CD90, CD73 and CD105 while did not express the surface markers for CD34, CD45 and HLA-DR. Also, we confirmed that hDMSCs possess the capacity to differentiate into multiple lineage including adipocyte, osteocyte, chondrocyte and precursor of hepatocyte lineage. Considering these results, we suggest that hDMSCs might be a valuable source of stem cells and could potentially be a useful source of clinical application. PMID:25163610

  3. Brain injuries in early foetal life: consequences for brain development.

    PubMed

    Mancini, J; Lethel, V; Hugonenq, C; Chabrol, B

    2001-01-01

    Learning disability and cerebral palsy are often related to factors present before birth. We report three patients (two with schizencephaly, one with unilateral cerebellar agenesis) in whom the timing of an insult to the foetus was known. In the first case, the mother had a trauma at 16 weeks of pregnancy and schizencephaly was discovered in the male infant associated with a left hemiplegia. In the second child, amniocentesis performed at 16 weeks into pregnancy may have been responsible for the same cortical anomaly. In the third case, sequential foetal echographies clearly demonstrated that an apparent unilateral cerebellar agenesis was related to an haemorrhagic event secondary to cerebellar trauma that occurred at 19 weeks of pregnancy. It is suggested that these brain malformations are related to an ischemic mechanism or a traumatic event in foetal life. PMID:11201424

  4. Transport across the rabbit foetal yolk-sac of fractions of IgG from several mammalian species.

    PubMed Central

    Hemmings, W A; Jones, R E; Faulk, W P

    1975-01-01

    Electrophoretically migrating fast and slow IgG fractions of rabbit, mouse, human, guinea-pig, dog, horse and cow sera were prepared by DEAE-cellulose chromatography, labelled with either 131I or 125I, and tested in passage across yolk-sac splanchnopleur of rabbits. In foetal sera, slow IgG generally reached higher concentrations than fast IgG, but even with mouse Ig, which has a high ratio between the two fractions, the difference was only four-fold. This differential between fast and slow IgG was not sufficient to explain the absorptive selectivity between IgG of different species. The capacity of isotopically labelled rabbit and equine IgG to be transported across the rabbit foetal yolk sac was also studied using fractions prepared by isoelectric focusing. Marked differences in concentration quotient (CQ) values (CQ equals (concentration in foetal serum)/(concentration in injected material)) between individual pI peaks for these species were found. Images FIG. 1 FIG. 2 PMID:1168622

  5. Targeting Androgen Receptor/Src Complex Impairs the Aggressive Phenotype of Human Fibrosarcoma Cells

    PubMed Central

    Di Donato, Marzia; Hayashi, Ryo; Arra, Claudio; Appella, Ettore; Auricchio, Ferdinando; Migliaccio, Antimo

    2013-01-01

    Background Hormones and growth factors influence the proliferation and invasiveness of human mesenchymal tumors. The highly aggressive human fibrosarcoma HT1080 cell line harbors classical androgen receptor (AR) that responds to androgens triggering cell migration in the absence of significant mitogenesis. As occurs in many human cancer cells, HT1080 cells also express epidermal growth factor receptor (EGFR). Experimental Findings: We report that the pure anti-androgen Casodex inhibits the growth of HT1080 cell xenografts in immune-depressed mice, revealing a novel role of AR in fibrosarcoma progression. In HT1080 cultured cells EGF, but not androgens, robustly increases DNA synthesis. Casodex abolishes the EGF mitogenic effect, implying a crosstalk between EGFR and AR. The mechanism underlying this crosstalk has been analyzed using an AR-derived small peptide, S1, which prevents AR/Src tyrosine kinase association and androgen-dependent Src activation. Present findings show that in HT1080 cells EGF induces AR/Src Association, and the S1 peptide abolishes both the assembly of this complex and Src activation. The S1 peptide inhibits EGF-stimulated DNA synthesis, cell matrix metalloproteinase-9 (MMP-9) secretion and invasiveness of HT1080 cells. Both Casodex and S1 peptide also prevent DNA synthesis and migration triggered by EGF in various human cancer-derived cells (prostate, breast, colon and pancreas) that express AR. Conclusion This study shows that targeting the AR domain involved in AR/Src association impairs EGF signaling in human fibrosarcoma HT1080 cells. The EGF-elicited processes inhibited by the peptide (DNA synthesis, MMP-9 secretion and invasiveness) cooperate in increasing the aggressive phenotype of HT1080 cells. Therefore, AR represents a new potential therapeutic target in human fibrosarcoma, as supported by Casodex inhibition of HT1080 cell xenografts. The extension of these findings in various human cancer-derived cell lines highlights the

  6. Comparison of foetal metabolic differentiation in three cattle muscles.

    PubMed

    Gagnière, H; Picard, B; Jurie, C; Geay, Y

    1999-01-01

    Metabolic differentiation of Semitendinosus (ST), Cutaneus trunci (CT) and Masseter (MA) in cattle foetuses aged from 110 to 260 days was studied by measuring isocitrate dehydrogenase (ICDH, oxidative) and lactate dehydrogenase (LDH, glycolytic) activities. The five LDH isoenzymes were separated by electrophoresis and assayed by densitometry. ICDH activity increased from 210 days onwards in the three muscles but more intensively in MA (oxidative). LDH activity increased from 170 days onwards in ST, 180 days onwards in CT and only from 210 days onwards in MA and was higher in the glycolytic muscles (ST and CT). The proportion of the LDH-M subunit increased during foetal life in glycolytic muscles. At 110 days, it was higher in CT, intermediate in ST and lower in MA. These results show that 1) metabolic differentiation of bovine muscle begins during the last third of foetal life and 2) the proportion of the LDH-M subunit seems to be related to the contractile type of adult muscle from the first stages of foetal life. PMID:10222501

  7. Claudin-20 promotes an aggressive phenotype in human breast cancer cells

    PubMed Central

    Martin, Tracey A; Lane, Jane; Ozupek, Hulya; Jiang, Wen G

    2013-01-01

    Claudin-20 is a member of the Claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Due to the increasing evidence supporting the role of TJ proteins in preventing tumor cell metastatic behavior, this study sought to evaluate the distribution of Claudin-20 in human breast cancer and the effect of Claudin-20 overexpression in human breast cancer cells. Q-PCR data from breast cancer primary tumors (n = 114) and matched background tissue (n = 30) showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p = 0.022). Following transformation of the breast cancer cell lines MDA-MB-231 and MCF7 with a Claudin-20 expression construct functional assays were performed to ascertain changes in cell behavior. Claudin-20 transformed cells showed significantly increased invasion (p < 0.005) and were significantly less adhesive than wild type cells (p < 0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Overexpression of Claudin-20 resulted in reduced transepithelial resistance (induced by the motogen HGF at 25 ng/ml, p = 0.0007). Interestingly, this was not mirrored by paracellular permeability, as overexpression of Claudin-20 caused a decrease in permeability. The introduction of Claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this Claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer. PMID:24665404

  8. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    PubMed Central

    Leyton-Mange, Jordan S.; Mills, Robert W.; Macri, Vincenzo S.; Jang, Min Young; Butte, Faraz N.; Ellinor, Patrick T.; Milan, David J.

    2014-01-01

    Summary In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  9. Rapid cellular phenotyping of human pluripotent stem cell-derived cardiomyocytes using a genetically encoded fluorescent voltage sensor.

    PubMed

    Leyton-Mange, Jordan S; Mills, Robert W; Macri, Vincenzo S; Jang, Min Young; Butte, Faraz N; Ellinor, Patrick T; Milan, David J

    2014-02-11

    In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  10. Nitric oxide induces cancer stem cell-like phenotypes in human lung cancer cells.

    PubMed

    Yongsanguanchai, Nuttida; Pongrakhananon, Varisa; Mutirangura, Apiwat; Rojanasakul, Yon; Chanvorachote, Pithi

    2015-01-15

    Even though tremendous advances have been made in the treatment of cancers during the past decades, the success rate among patients with cancer is still dismal, largely because of problems associated with chemo/radioresistance and relapse. Emerging evidence has indicated that cancer stem cells (CSCs) are behind the resistance and recurrence problems, but our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. Here we show how nitric oxide (NO), a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human non-small cell lung carcinoma H292 and H460 cells. Exposure of NO gradually altered the cell morphology toward mesenchymal stem-like shape. NO exposure promoted CSC-like phenotype, indicated by increased expression of known CSC markers, CD133 and ALDH1A1, in the exposed cells. These effects of NO on stemness were reversible after cessation of the NO treatment for 7 days. Furthermore, such effect was reproducible using another NO donor, S-nitroso-N-acetylpenicillamine. Importantly, inhibition of NO by the known NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5 tetramethylimidazoline-1-oxy-3-oxide strongly inhibited CSC-like aggressive cellular behavior and marker expression. Last, we unveiled the underlying mechanism of NO action through the activation of caveolin-1 (Cav-1), which is upregulated by NO and is responsible for the aggressive behavior of the cells, including anoikis resistance, anchorage-independent cell growth, and increased cell migration and invasion. These findings indicate a novel role of NO in CSC regulation and its importance in aggressive cancer behaviors through Cav-1 upregulation. PMID:25411331

  11. Iron induces cancer stem cells and aggressive phenotypes in human lung cancer cells.

    PubMed

    Chanvorachote, Pithi; Luanpitpong, Sudjit

    2016-05-01

    Evidence has accumulated in support of the critical impact of cancer stem cells (CSCs) behind the chemotherapeutic failure, cancer metastasis, and subsequent disease recurrence and relapse, but knowledge of how CSCs are regulated is still limited. Redox status of the cells has been shown to dramatically influence cell signaling and CSC-like aggressive behaviors. Here, we investigated how subtoxic concentrations of iron, which have been found to specifically induce cellular hydroxyl radical, affected CSC-like subpopulations of human non-small cell lung carcinoma (NSCLC). We reveal for the first time that subchronic iron exposure and higher levels of hydroxyl radical correlated well with increased CSC-like phenotypes. The iron-exposed NSCLC H460 and H292 cells exhibited a remarkable increase in propensities to form CSC spheroids and to proliferate, migrate, and invade in parallel with an increase in level of a well-known CSC marker, ABCG2. We further observed that such phenotypic changes induced by iron were not related to an epithelial-to-mesenchymal transition (EMT). Instead, the sex-determining region Y (SRY)-box 9 protein (SOX9) was substantially linked to iron treatment and hydroxyl radical level. Using gene manipulations, including ectopic SOX9 overexpression and SOX9 short hairpin RNA knockdown, we have verified that SOX9 is responsible for CSC enrichment mediated by iron. These findings indicate a novel role of iron via hydroxyl radical in CSC regulation and its importance in aggressive cancer behaviors and likely metastasis through SOX9 upregulation. PMID:26911281

  12. CULTURE CONDITIONS PROFOUNDLY IMPACT PHENOTYPE IN BEAS-2B, A HUMAN PULMONARY EPITHELIAL MODEL

    PubMed Central

    Zhao, Fei; Klimecki, Walter T.

    2015-01-01

    BEAS-2B, an immortalized, human lung epithelial cell line, has been used to model pulmonary epithelial function for over 30 years. The BEAS-2B phenotype can be modulated by culture conditions that include the presence or absence of fetal bovine serum (FBS). The popularity of BEAS-2B as a model of arsenic toxicology, and the common use of BEAS-2B cultured both with and without FBS, led us to investigate the impact of FBS on BEAS-2B in the context of arsenic toxicology. Comparison of genome-wide gene expression in BEAS-2B cultured with or without FBS revealed altered expression in several biological pathways, including those related to carcinogenesis and energy metabolism. Real-time measurements of oxygen consumption and glycolysis in BEAS-2B demonstrated that FBS culture conditions were associated with a 1.4-fold increase in total glycolytic capacity, a 1.9-fold increase in basal respiration, a 2.0-fold increase in oxygen consumed for ATP production, and a 2.8-fold increase in maximal respiration, compared to BEAS-2B cultured without FBS. Comparisons of the transcriptome changes in BEAS-2B resulting from FBS exposure to the transcriptome changes resulting from exposure to 1 μM sodium arsenite revealed that mRNA levels of 43% of the arsenite-modulated genes were also modulated by FBS. Cytotoxicity studies revealed that BEAS-2B cells exposed to 5% FBS for 8 weeks were almost 5 times more sensitive to arsenite cytotoxicity than non-FBS-exposed BEAS-2B cells. Phenotype changes induced in BEAS-2B by FBS suggest that culture conditions should be carefully considered when using BEAS-2B as an experimental model of arsenic toxicity. PMID:25524072

  13. Lack of prolidase causes a bone phenotype both in human and in mouse.

    PubMed

    Besio, Roberta; Maruelli, Silvia; Gioia, Roberta; Villa, Isabella; Grabowski, Peter; Gallagher, Orla; Bishop, Nicholas J; Foster, Sarah; De Lorenzi, Ersilia; Colombo, Raffaella; Diaz, Josè Luis Dapena; Moore-Barton, Haether; Deshpande, Charu; Aydin, Halil Ibrahim; Tokatli, Aysegul; Kwiek, Bartlomiej; Kasapkara, Cigdem Seher; Adisen, Esra Ozsoy; Gurer, Mehmet Ali; Di Rocco, Maja; Phang, James M; Gunn, Teresa M; Tenni, Ruggero; Rossi, Antonio; Forlino, Antonella

    2015-03-01

    The degradation of the main fibrillar collagens, collagens I and II, is a crucial process for skeletal development. The most abundant dipeptides generated from the catabolism of collagens contain proline and hydroxyproline. In humans, prolidase is the only enzyme able to hydrolyze dipeptides containing these amino acids at their C-terminal end, thus being a key player in collagen synthesis and turnover. Mutations in the prolidase gene cause prolidase deficiency (PD), a rare recessive disorder. Here we describe 12 PD patients, 9 of whom were molecularly characterized in this study. Following a retrospective analysis of all of them a skeletal phenotype associated with short stature, hypertelorism, nose abnormalities, microcephaly, osteopenia and genu valgum, independent of both the type of mutation and the presence of the mutant protein was identified. In order to understand the molecular basis of the bone phenotype associated with PD, we analyzed a recently identified mouse model for the disease, the dark-like (dal) mutant. The dal/dal mice showed a short snout, they were smaller than controls, their femurs were significantly shorter and pQCT and μCT analyses of long bones revealed compromised bone properties at the cortical and at the trabecular level in both male and female animals. The differences were more pronounce at 1 month being the most parameters normalized by 2 months of age. A delay in the formation of the second ossification center was evident at postnatal day 10. Our work reveals that reduced bone growth was due to impaired chondrocyte proliferation and increased apoptosis rate in the proliferative zone associated with reduced hyperthrophic zone height. These data suggest that lack of prolidase, a cytosolic enzyme involved in the final stage of protein catabolism, is required for normal skeletogenesis especially at early age when the requirement for collagen synthesis and degradation is the highest. PMID:25460580

  14. CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages

    PubMed Central

    Fergusson, Joannah R.; Smith, Kira E.; Fleming, Vicki M.; Rajoriya, Neil; Newell, Evan W.; Simmons, Ruth; Marchi, Emanuele; Björkander, Sophia; Kang, Yu-Hoi; Swadling, Leo; Kurioka, Ayako; Sahgal, Natasha; Lockstone, Helen; Baban, Dilair; Freeman, Gordon J.; Sverremark-Ekström, Eva; Davis, Mark M.; Davenport, Miles P.; Venturi, Vanessa; Ussher, James E.; Willberg, Christian B.; Klenerman, Paul

    2014-01-01

    The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a novel population known as Mucosal Associated Invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, that can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Further, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161 expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both TCR expression and cell lineage. PMID:25437561

  15. Cultures of human tracheal gland cells of mucous or serous phenotype

    PubMed Central

    Finkbeiner, Walter E.; Zlock, Lorna T.; Mehdi, Irum

    2009-01-01

    There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced “mucous” cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced “serous” cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both “serous” and “mucous” cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl. PMID:19998060

  16. Dilute passage promotes expression of genetic and phenotypic variants of human immunodeficiency virus type 1 in cell culture.

    PubMed Central

    Sánchez-Palomino, S; Rojas, J M; Martínez, M A; Fenyö, E M; Nájera, R; Domingo, E; López-Galíndez, C

    1993-01-01

    We have studied the extent of genetic and phenotypic diversification of human immunodeficiency virus type 1 (HIV-1) upon 15 serial passages of clonal viral populations in MT-4 cell cultures. Several genetic and phenotypic modifications previously noted during evolution of HIV-1 in infected humans were also observed upon passages of the virus in cell culture. Notably, the transition from non-syncytium-inducing to syncytium-inducing phenotype (previously observed during disease progression) and fixation of amino acid substitutions at the main antigenic loop V3 of gp120 were observed in the course of replication of the virus in MT-4 cell cultures in the absence of immune selection. Interestingly, most genetic and phenotypic alterations occurred upon passage of the virus at a low multiplicity of infection (0.001 infectious particles per cell) rather than at a higher multiplicity of infection (0.1 infectious particles per cell). The degree of genetic diversification attained by HIV-1, estimated by the RNase A mismatch cleavage method and by nucleotide sequencing, is of about 0.03% of genomic sites mutated after 15 serial passages. This value is not significantly different from previous estimates for foot-and-mouth disease virus when subjected to a similar process and analysis. We conclude that several genetic and phenotypic modifications of HIV-1 previously observed in vivo occur also in the constant environment provided by a cell culture system. Dilute passage promotes in a highly significant way the expression of deviant HIV-1 genomes. Images PMID:8474182

  17. A matter of identity - Phenotype and differentiation potential of human somatic stem cells.

    PubMed

    New, S E P; Alvarez-Gonzalez, C; Vagaska, B; Gomez, S G; Bulstrode, N W; Madrigal, A; Ferretti, P

    2015-07-01

    Human somatic stem cells with neural differentiation potential can be valuable for developing cell-based therapies, including treatment of birth-related defects, while avoiding issues associated with cell reprogramming. Precisely defining the "identity" and differentiation potential of somatic stem cells from different sources, has proven difficult, given differences in sets of specific markers, protocols used and lack of side-by-side characterization of these cells in different studies. Therefore, we set to compare expression of mesenchymal and neural markers in human umbilical cord-derived mesenchymal stem cells (UC-MSCs), pediatric adipose-derived stem cells (p-ADSCs) in parallel with human neural stem cells (NSCs). We show that UC-MSCs at a basal level express mesenchymal and so-called "neural" markers, similar to that we previously reported for the p-ADSCs. All somatic stem cell populations studied, independently from tissue and patient of origin, displayed a remarkably similar expression of surface markers, with the main difference being the restricted expression of CD133 and CD34 to NSCs. Expression of certain surface and neural markers was affected by the expansion medium used. As predicted, UC-MSCs and p-ADSCs demonstrated tri-mesenchymal lineage differentiation potential, though p-ADSCs display superior chondrogenic differentiation capability. UC-MSCs and p-ADSCs responded also to neurogenic induction by up-regulating neuronal markers, but crucially they appeared morphologically immature when compared with differentiated NSCs. This highlights the need for further investigation into the use of these cells for neural therapies. Crucially, this study demonstrates the lack of simple means to distinguish between different cell types and the effect of culture conditions on their phenotype, and indicates that a more extensive set of markers should be used for somatic stem cell characterization, especially when developing therapeutic approaches. PMID:25957945

  18. Exploiting CpG hypermutability to identify phenotypically significant variation within human protein-coding genes.

    PubMed

    Ying, Hua; Huttley, Gavin

    2011-01-01

    The CpG dinucleotide is disproportionately represented in human genetic variation due to the hypermutability of 5-methyl-cytosine (5mC). We exploit this hypermutability and a novel codon substitution model to identify candidate functionally important exonic nucleotides. Population genetic theory suggests that codon positions with high cross-species CpG frequency will derive from stronger purifying selection. Using the phylogeny-based maximum likelihood inference framework, we applied codon substitution models with context-dependent parameters to measure the mutagenic and selective processes affecting CpG dinucleotides within exonic sequence. The suitability of these models was validated on >2,000 protein coding genes from a naturally occurring biological control, four yeast species that do not methylate their DNA. As expected, our analyses of yeast revealed no evidence for an elevated CpG transition rate or for substitution suppression affecting CpG-containing codons. Our analyses of >12,000 protein-coding genes from four primate lineages confirm the systemic influence of 5mC hypermutability on the divergence of these genes. After adjusting for confounding influences of mutation and the properties of the encoded amino acids, we confirmed that CpG-containing codons are under greater purifying selection in primates. Genes with significant evidence of enhanced suppression of nonsynonymous CpG changes were also shown to be significantly enriched in Online Mendelian Inheritance in Man. We developed a method for ranking candidate phenotypically influential CpG positions in human genes. Application of this method indicates that of the ∼1 million exonic CpG dinucleotides within humans, ∼20% are strong candidates for both hypermutability and disease association. PMID:21398426

  19. Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells

    PubMed Central

    Tian, Lipeng; Deshmukh, Abhijeet; Prasad, Neha; Jang, Yoon-Young

    2016-01-01

    Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults. PMID:27570479

  20. Alcohol Increases Liver Progenitor Populations and Induces Disease Phenotypes in Human IPSC-Derived Mature Stage Hepatic Cells.

    PubMed

    Tian, Lipeng; Deshmukh, Abhijeet; Prasad, Neha; Jang, Yoon-Young

    2016-01-01

    Alcohol consumption has long been a global problem affecting human health, and has been found to influence both fetal and adult liver functions. However, how alcohol affects human liver development and liver progenitor cells remains largely unknown. Here, we used human induced pluripotent stem cells (iPSCs) as a model to examine the effects of alcohol, on multi-stage hepatic cells including hepatic progenitors, early and mature hepatocyte-like cells derived from human iPSCs. While alcohol has little effect on endoderm development from iPSCs, it reduces formation of hepatic progenitor cells during early hepatic specification. The proliferative activities of early and mature hepatocyte-like cells are significantly decreased after alcohol exposure. Importantly, at a mature stage of hepatocyte-like cells, alcohol treatment increases two liver progenitor subsets, causes oxidative mitochondrial injury and results in liver disease phenotypes (i.e., steatosis and hepatocellular carcinoma associated markers) in a dose dependent manner. Some of the phenotypes were significantly improved by antioxidant treatment. This report suggests that fetal alcohol exposure may impair generation of hepatic progenitors at early stage of hepatic specification and decrease proliferation of fetal hepatocytes; meanwhile alcohol injury in post-natal or mature stage human liver may contribute to disease phenotypes. This human iPSC model of alcohol-induced liver injury can be highly valuable for investigating alcoholic injury in the fetus as well as understanding the pathogenesis and ultimately developing effective treatment for alcoholic liver disease in adults. PMID:27570479

  1. Fludarabine phosphate selectively inhibits growth and modifies the antigenic phenotype of human glioblastoma-multiforme cells expressing a multidrug resistance phenotype.

    PubMed

    Jiang, H; Su, Z; Datta, S; Guarini, L; Waxman, S; Fisher, P

    1992-07-01

    Fludarabine phosphate (FLU), the 2-fluro derivative of Ara-A, 9-beta-D-arabino-furanosyl-2-fluoroadenine, has been shown to display both in vitro and in vivo antiproliferative activity toward a variety of murine tumors and human lymphoid malignancies. In the present study, we have determined the effect of FLU, alone and in combination with recombinant human fibroblast interferon (IFN-B), on in vitro growth, gene expression and the antigenic phenotype of human glioblastoma multiforme (GBM) cells displaying a multidrug sensitive and a multidrug resistant (MDR) phenotype. FLU exhibited a marked differential toxicity toward GBM-MDR cells versus the multidrug sensitive GBM parental cell line. Growth of GBM-MDR cells for seven days in 2.5 to 7.5 muM FLU resulted in a dose-dependent reduction or elimination of growth which persisted after removal of this agent. In contrast, recovery from FLU-induced growth suppression was observed in parental multidrug sensitive GBM cells. Acquisition of increased FLU sensitivity in GBM-MDR cells did not appear to result from selection for a subset of sensitive cells or an artifact associated with the DNA-transfection process. This conclusion is supported by the similar pattern of FLU resistance in GBM-18 clones isolated after transfection with a cloned hygromycin resistance gene and selection for resistance to hygromycin. The antiproliferative and toxic effect of FLU was increased in GBM-MDR cells by simultaneous growth in IFN-B and the toxic effect of FLU could be blocked in a dose-dependent manner by the simultaneous addition of deoxycytidine. In contrast, the toxicity of FLU toward GBM-MDR cells was not altered when cells were grown in the presence or absence of colchicine or by the administration of verapamil, which can reverse the MDR phenotype in GBM-MDR cells. The selective toxicity of FLU toward GBM-MDR versus GBM-18 cells was not associated with a consistent differential change in all of the GBM-18 MDR clones in the steady

  2. The more g-loaded, the more heritable, evolvable, and phenotypically variable: Homology with humans in chimpanzee cognitive abilities

    PubMed Central

    Hopkins, William D.

    2015-01-01

    Expanding on a recent study that identified a heritable general intelligence factor (g) among individual chimpanzees from a battery of cognitive tasks, we hypothesized that the cognitive abilities that are more g-loaded would be more heritable and would present more additive genetic variance, in addition to showing more phenotypic variability. This pattern was confirmed, and is comparable to that found in humans, indicating fundamental homology. Finally, tool use presented the highest heritability, the largest amount of additive genetic variance and of phenotypic variance, consistent with previous findings indicating that it is associated with high interspecies variance and evolutionary rates in comparative primate studies. PMID:26005227

  3. The impact of cis-acting polymorphisms on the human phenotype.

    PubMed

    Jones, Bryony L; Swallow, Dallas M

    2011-12-01

    Cis-acting polymorphisms that affect gene expression are now known to be frequent, although the extent and mechanisms by which such variation affects the human phenotype are, as yet, only poorly understood. Key signatures of cis-acting variation are differences in gene expression that are tightly associated with regulatory SNPs or expression Quantitative Trait Loci (eQTL) and an imbalance of allelic expression (AEI) in heterozygous samples. Such cis-acting sequence differences appear often to have been under selection within and between populations and are also thought to be important in speciation. Here we describe the example of lactase persistence. In medical research, variants that affect regulation in cis have been implicated in both monogenic and polygenic disorders, and in the metabolism of drugs. In this review we suggest that by further understanding common regulatory variations and how they interact with other genetic and environmental variables it will be possible to gain insight into important mechanisms behind complex disease, with the potential to lead to new methods of diagnosis and treatments. PMID:23205161

  4. Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

    PubMed Central

    Kondo, Masahiro; Yamaoka, Kunihiro; Tanaka, Yoshiya

    2014-01-01

    An inflammatory milieu breaks down the cartilage matrix and induces chondrocyte apoptosis, resulting in cartilage destruction in patients with cartilage degenerative diseases, such as rheumatoid arthritis or osteoarthritis. Because of the limited regenerative ability of chondrocytes, defects in cartilage are irreversible and difficult to repair. Mesenchymal stem cells (MSCs) are expected to be a new tool for cartilage repair because they are present in the cartilage and are able to differentiate into multiple lineages of cells, including chondrocytes. Although clinical trials using MSCs for patients with cartilage defects have already begun, its efficacy and repair mechanisms remain unknown. A PubMed search conducted in October 2014 using the following medical subject headings (MeSH) terms: mesenchymal stromal cells, chondrogenesis, and cytokines resulted in 204 articles. The titles and abstracts were screened and nine articles relevant to “inflammatory” cytokines and “human” MSCs were identified. Herein, we review the cell biology and mechanisms of chondrocyte phenotype acquisition from human MSCs in an inflammatory milieu and discuss the clinical potential of MSCs for cartilage repair. PMID:25407530

  5. Gene Expression Profiling of Human Decidual Macrophages: Evidence for Immunosuppressive Phenotype

    PubMed Central

    Gustafsson, Charlotte; Mjösberg, Jenny; Matussek, Andreas; Geffers, Robert; Matthiesen, Leif; Berg, Göran; Sharma, Surendra; Buer, Jan; Ernerudh, Jan

    2008-01-01

    Background Although uterine macrophages are thought to play an important regulatory role at the maternal-fetal interface, their global gene expression profile is not known. Methodology/Principal Findings Using micro-array comprising approximately 14,000 genes, the gene expression pattern of human first trimester decidual CD14+ monocytes/macrophages was characterized and compared with the expression profile of the corresponding cells in blood. Some of the key findings were confirmed by real time PCR or by secreted protein. A unique gene expression pattern intrinsic of first trimester decidual CD14+ cells was demonstrated. A large number of regulated genes were functionally related to immunomodulation and tissue remodelling, corroborating polarization patterns of differentiated macrophages mainly of the alternatively activated M2 phenotype. These include known M2 markers such as CCL-18, CD209, insulin-like growth factor (IGF)-1, mannose receptor c type (MRC)-1 and fibronectin-1. Further, the selective up-regulation of triggering receptor expressed on myeloid cells (TREM)-2, alpha-2-macroglobulin (A2M) and prostaglandin D2 synthase (PGDS) provides new insights into the regulatory function of decidual macrophages in pregnancy that may have implications in pregnancy complications. Conclusions/Significance The molecular characterization of decidual macrophages presents a unique transcriptional profile replete with important components for fetal immunoprotection and provides several clues for further studies of these cells. PMID:18446208

  6. Embryonic Zebrafish: Different Phenotypes after Injection of Human Uveal Melanoma Cells.

    PubMed

    van der Ent, Wietske; Burrello, Claudia; de Lange, Mark J; van der Velden, Pieter A; Jochemsen, Aart G; Jager, Martine J; Snaar-Jagalska, B Ewa

    2015-04-01

    Although murine xenograft models for human uveal melanoma (UM) are available, they are of limited utility for screening large compound libraries for the discovery of new drugs. We need new preclinical models which can efficiently evaluate drugs that can treat UM metastases. The zebrafish embryonic model is ideal for drug screening purposes because it allows the investigation of potential antitumor properties of drugs within 1 week. The optical transparency of the zebrafish provides unique possibilities for live imaging of fluorescence-labelled cancer cells and their behavior. In addition, the adaptive immune response, which is responsible for the rejection of transplanted material, is not yet present in the early stages of fish development, and systemic immunosuppression is therefore not required to allow growth of tumor cells. We studied the behavior of UM cells following injection into zebrafish embryos and observed different phenotypes. We also analyzed cell migration, proliferation, formation of micrometastasis and interaction with the host microenvironment. Significant differences were noted between cell lines: cells derived from metastases showed more migration and proliferation than cells derived from the primary tumors. The addition of the c-Met inhibitor crizotinib to the water in which the larvae were kept reduced the migration and proliferation of UM cells expressing c-Met. This indicates the applicability of the zebrafish xenografts for testing novel inhibitory compounds and provides a fast and sensitive in vivo vertebrate model for preclinical drug screening to combat UM. PMID:27171126

  7. The Phenotypic and Functional Features of Human M2 Macrophages Generated Under Low Serum Conditions.

    PubMed

    Sakhno, L V; Shevela, E Ya; Tikhonova, M A; Ostanin, A A; Chernykh, E R

    2016-02-01

    The phenotypic and functional features of human M2 macrophages, in particular, their immunosuppressive activity, can considerably vary depending on M2 polarizing stimulus. This study was aimed at the investigation of cytokine production and pro-apoptogenic/inhibitory molecule expression in macrophages generated with GM-CSF using either standard conditions (M1) or deficiency of serum/growth factors (M2-LS cells). In contrast to M1, M2-LS cells were characterized by an enhanced content of CD206(+), B7-H1(+), FasL(+) and TRAIL(+) cells along with a decreased production of IFN-γ, IL-5, IL-6, IL-13, TNF-α, IL-17 and MCP-1. In addition, M2-LS exhibited a lower T cell stimulatory activity in MLC that was associated with the higher numbers of apoptotic and the lower numbers of proliferating T cells. B7-H1 plays a key role in M2-LS-mediated cytotoxic effects as the neutralization of B7-H1 reduces the apoptosis-inducing activity of M2-LS, while the blocking of CD206 and TRAIL reduces the cytostatic activity of M2 macrophages. PMID:26678544

  8. Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype.

    PubMed

    Camats, Núria; Fernández-Cancio, Mónica; Audí, Laura; Mullis, Primus E; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E

    2015-01-01

    MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life. PMID:26580071

  9. Humans display a 'cooperative phenotype' that is domain general and temporally stable.

    PubMed

    Peysakhovich, Alexander; Nowak, Martin A; Rand, David G

    2014-01-01

    Understanding human cooperation is of major interest across the natural and social sciences. But it is unclear to what extent cooperation is actually a general concept. Most research on cooperation has implicitly assumed that a person's behaviour in one cooperative context is related to their behaviour in other settings, and at later times. However, there is little empirical evidence in support of this assumption. Here, we provide such evidence by collecting thousands of game decisions from over 1,400 individuals. A person's decisions in different cooperation games are correlated, as are those decisions and both self-report and real-effort measures of cooperation in non-game contexts. Equally strong correlations exist between cooperative decisions made an average of 124 days apart. Importantly, we find that cooperation is not correlated with norm-enforcing punishment or non-competitiveness. We conclude that there is a domain-general and temporally stable inclination towards paying costs to benefit others, which we dub the 'cooperative phenotype'. PMID:25225950

  10. Mutations in collagen 18A1 and their relevance to the human phenotype.

    PubMed

    Passos-Bueno, Maria Rita; Suzuki, Oscar T; Armelin-Correa, Lucia M; Sertié, Andréa L; Errera, Flavia I V; Bagatini, Kelly; Kok, Fernando; Leite, Katia R M

    2006-03-01

    Collagen XVIII, a proteoglycan, is a component of basement membranes (BMs). There are three distinct isoforms that differ only by their N-terminal, but with a specific pattern of tissue and developmental expression. Cleavage of its C-terminal produces endostatin, an inhibitor of angiogenesis. In its N-terminal, there is a frizzled motif which seems to be involved in Wnt signaling. Mutations in this gene cause Knobloch syndrome KS), an autosomal recessive disorder characterized by vitreoretinal and macular degeneration and occipital encephalocele. This review discusses the effect of both rare and polymorphic alleles in the human phenotype, showing that deficiency of one of the collagen XVIII isoforms is sufficient to cause KS and that null alleles causing deficiency of all collagen XVIII isoforms are associated with a more severe ocular defect. This review besides illustrating the functional importance of collagen XVIII in eye development and its structure maintenance throughout life, it also shows its role in other tissues and organs, such as nervous system and kidney. PMID:16532212

  11. Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

    PubMed Central

    Golda, Adam; Jurecka, Agnieszka; Gajda, Karolina; Tylki-Szymańska, Anna; Lalik, Anna

    2015-01-01

    Background Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. Aim The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). Results Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 μg/mL DS and reduction at 0 and 100 μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. Conclusion Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors. PMID:26937388

  12. Human MAMLD1 Gene Variations Seem Not Sufficient to Explain a 46,XY DSD Phenotype

    PubMed Central

    Audí, Laura; Mullis, Primus E.; Moreno, Francisca; González Casado, Isabel; López-Siguero, Juan Pedro; Corripio, Raquel; Bermúdez de la Vega, José Antonio; Blanco, José Antonio; Flück, Christa E.

    2015-01-01

    MAMLD1 is thought to cause disordered sex development in 46,XY patients. But its role is controversial because some MAMLD1 variants are also detected in normal individuals, several MAMLD1 mutations have wild-type activity in functional tests, and the male Mamld1-knockout mouse has normal genitalia and reproduction. Our aim was to search for MAMLD1 variations in 108 46,XY patients with disordered sex development, and to test them functionally. We detected MAMDL1 variations and compared SNP frequencies in controls and patients. We tested MAMLD1 transcriptional activity on promoters involved in sex development and assessed the effect of MAMLD1 on androgen production. MAMLD1 expression in normal steroid-producing tissues and mutant MAMLD1 protein expression were also assessed. Nine MAMLD1 mutations (7 novel) were characterized. In vitro, most MAMLD1 variants acted similarly to wild type. Only the L210X mutation showed loss of function in all tests. We detected no effect of wild-type or MAMLD1 variants on CYP17A1 enzyme activity in our cell experiments, and Western blots revealed no significant differences for MAMLD1 protein expression. MAMLD1 was expressed in human adult testes and adrenals. In conclusion, our data support the notion that MAMLD1 sequence variations may not suffice to explain the phenotype in carriers and that MAMLD1 may also have a role in adult life. PMID:26580071

  13. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington’s Disease T Lymphocytes

    PubMed Central

    Miller, James R. C.; Träger, Ulrike; Andre, Ralph; Tabrizi, Sarah J.

    2015-01-01

    Huntington’s disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington’s disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington’s disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington’s disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington’s disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington’s disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington’s disease innate immune system should not be extended to include the adaptive immune system. PMID:26529236

  14. Phenotypic Characterization and ln Vivo Localization of Human Adipose-Derived Mesenchymal Stem Cells

    PubMed Central

    Ryu, Young-Joon; Cho, Tae-Jun; Lee, Dong-Sup; Choi, Jin-Young; Cho, Jaejin

    2013-01-01

    Human adipose-derived mesenchymal stem cells (hADMSCs) are a potential cell source for autologous cell therapy due to their regenerative ability. However, detailed cytological or phenotypic characteristics of these cells are still unclear. Therefore, we determined and compared cell size, morphology, ultrastructure, and immunohistochemical (IHC) expression profiles of isolated hADMSCs and cells located in human adipose tissues. We also characterized the localization of these cells in vivo. Light microscopy examination at low power revealed that hADMSCs acquired a spindle-shaped morphology after four passages. Additionally, high power views showed that these cells had various sizes, nuclear contours, and cytoplasmic textures. To further evaluate cell morphology, transmission electron microscopy was performed. hADMSCs typically had ultrastructural characteristics similar to those of primitive mesenchymal cells including a relatively high nuclear/cytosol ratio, prominent nucleoli, immature cytoplasmic organelles, and numerous filipodia. Some cells contained various numbers of lamellar bodies and lipid droplets. IHC staining demonstrated that PDGFR and CD10 were constitutively expressed in most hADMSCs regardless of passage number but expression levels of α-SMA, CD68, Oct4 and c-kit varied. IHC staining of adipose tissue showed that cells with immunophenotypic characteristics identical to those of hADMSCs were located mainly in the perivascular adventitia not in smooth muscle area. In summary, hADMSCs were found to represent a heterogeneous cell population with primitive mesenchymal cells that were mainly found in the perivascular adventitia. Furthermore, the cell surface markers would be CD10/PDGFR. To obtain defined cell populations for therapeutic purposes, further studies will be required to establish more specific isolation methods. PMID:23677376

  15. Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes

    PubMed Central

    Pinnix, Chelsea C.; Lee, John T.; Liu, Zhao-Jun; McDaid, Ronan; Balint, Klara; Beverly, Levi J.; Brafford, Patricia A.; Xiao, Min; Himes, Benjamin; Zabierowski, Susan E.; Yashiro-Ohtani, Yumi; Nathanson, Katherine L.; Bengston, Ana; Pollock, Pamela M.; Weeraratna, Ashani T.; Nickoloff, Brian J.; Pear, Warren S.; Capobianco, Anthony J.; Herlyn, Meenhard

    2009-01-01

    The importance of MAPK signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf; yet, clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions demonstrated that Notch activity is significantly higher in melanomas than their non-transformed counterparts. The use of a constitutively-active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a ‘driving’ event in melanocytic transformation or instead a ‘passenger’ event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth suggesting that Notch, alone, is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene; this new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease. PMID:19549918

  16. [A girl with foetal valproate syndrome and autism spectrum disorder].

    PubMed

    Solignac, I

    2016-01-01

    We study the case of a 12-year-old girl who, following intra-uterine exposure to valproate, was diagnosed with foetal valproate syndrome, characterised at birth by dysmorphic features. The use of valproate during pregnancy (because of epilepsy or bipolar disorder) can cause not only structural defects in the growing foetus, but also problems in cognitive development and in adaptive and emotional/behavioural functioning in later life. We evaluate these domains of development in our discussion and suggest several other drugs, less harmful than valproate, which can be used to treat epilepsy or bipolar disorder during pregnancy. PMID:26979855

  17. [Need for extensive education when implementing new foetal monitoring technology].

    PubMed

    Colov, Nina S Palmgren

    2007-09-24

    ST-waveform analysis of the foetal electrocardiogram (STAN) has been shown to reduce the frequency of metabolic acidosis in newborns and to reduce the rate of caesarean sections. However, STAN technology requires an extensive educational effort during the initial implementation with certification of all midwives and doctors involved. This study shows that the positive results of using STAN could be reproduced over a five-year-period after completion of the initial teaching effort and certification of personnel and when STAN was part of everyday life at the labour ward at Gentofte Hospital. PMID:17953889

  18. Foetal Antiepileptic Drug Exposure and Verbal versus Non-Verbal Abilities at Three Years of Age

    ERIC Educational Resources Information Center

    Meador, Kimford J.; Baker, Gus A.; Browning, Nancy; Cohen, Morris J.; Clayton-Smith, Jill; Kalayjian, Laura A.; Kanner, Andres; Liporace, Joyce D.; Pennell, Page B.; Privitera, Michael; Loring, David W.

    2011-01-01

    We previously reported that foetal valproate exposure impairs intelligence quotient. In this follow-up investigation, we examined dose-related effects of foetal antiepileptic drug exposure on verbal and non-verbal cognitive measures. This investigation is an ongoing prospective observational multi-centre study in the USA and UK, which has enrolled…

  19. Highly efficient transduction of human plasmacytoid dendritic cells without phenotypic and functional maturation

    PubMed Central

    Veron, Philippe; Boutin, Sylvie; Martin, Samia; Chaperot, Laurence; Plumas, Joel; Davoust, Jean; Masurier, Carole

    2009-01-01

    Background Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1α) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols. PMID:19173717

  20. In Situ Characterizing Membrane Lipid Phenotype of Human Lung Cancer Cell Lines Using Mass Spectrometry Profiling

    PubMed Central

    He, Manwen; Guo, Shuai; Ren, Junling; Li, Zhili

    2016-01-01

    Abnormal lipid metabolisms are closely associated with cancers. In this study, mass spectrometry was employed to in situ investigate the associations of membrane lipid phenotypes of six human lung cancer cell lines (i.e., A549, H1650, H1975 from adenocarcinoma, H157 and H1703 from squamous cell carcinomas, and H460 from a large cell carcinoma) with cancer cell types and finally total 230 lipids were detected. Based these 230 lipids, partial least-square discriminant analysis indicated that fifteen lipids (i.e., PE 18:0_18:1, PI 18:0_20:4, SM 42:2, PE 16:0_20:4, PE 36:2, PC 36:2, SM 34:1, PA 38:3,C18:0, C22:4, PA 34:2, C20:5, C20:2, C18:2, and CerP 36:2) with variable importance in the projection (VIP) value of > 1.0 could be used to differentiate six cancer cell lines with the Predicted Residual Sum of Square (PRESS) score of 0.1974. Positive correlation between polyunsaturated fatty acids (i.e., C20:4, C22:4, C22:5, and C22:6) and polyunsaturated phospholipids (PE 16:0_20:4, PE 38:4, and PI 18:0_20:4) was observed in lung adenocarcinoma cells, especially for H1975 cells. Three adenocarcinoma cell lines (i.e., A549, H1650, and H1975) could be differentiated from other lung cancer cell lines based on the expression of C18:1, C20:1, C20:2, C20:5, and C22:6. PMID:27162539

  1. In Situ Characterizing Membrane Lipid Phenotype of Human Lung Cancer Cell Lines Using Mass Spectrometry Profiling.

    PubMed

    He, Manwen; Guo, Shuai; Ren, Junling; Li, Zhili

    2016-01-01

    Abnormal lipid metabolisms are closely associated with cancers. In this study, mass spectrometry was employed to in situ investigate the associations of membrane lipid phenotypes of six human lung cancer cell lines (i.e., A549, H1650, H1975 from adenocarcinoma, H157 and H1703 from squamous cell carcinomas, and H460 from a large cell carcinoma) with cancer cell types and finally total 230 lipids were detected. Based these 230 lipids, partial least-square discriminant analysis indicated that fifteen lipids (i.e., PE 18:0_18:1, PI 18:0_20:4, SM 42:2, PE 16:0_20:4, PE 36:2, PC 36:2, SM 34:1, PA 38:3,C18:0, C22:4, PA 34:2, C20:5, C20:2, C18:2, and CerP 36:2) with variable importance in the projection (VIP) value of > 1.0 could be used to differentiate six cancer cell lines with the Predicted Residual Sum of Square (PRESS) score of 0.1974. Positive correlation between polyunsaturated fatty acids (i.e., C20:4, C22:4, C22:5, and C22:6) and polyunsaturated phospholipids (PE 16:0_20:4, PE 38:4, and PI 18:0_20:4) was observed in lung adenocarcinoma cells, especially for H1975 cells. Three adenocarcinoma cell lines (i.e., A549, H1650, and H1975) could be differentiated from other lung cancer cell lines based on the expression of C18:1, C20:1, C20:2, C20:5, and C22:6. PMID:27162539

  2. Study of the Aminoglycoside Subsistence Phenotype of Bacteria Residing in the Gut of Humans and Zoo Animals

    PubMed Central

    Bello González, Teresita de J.; Zuidema, Tina; Bor, Gerrit; Smidt, Hauke; van Passel, Mark W. J.

    2016-01-01

    Recent studies indicate that next to antibiotic resistance, bacteria are able to subsist on antibiotics as a carbon source. Here we evaluated the potential of gut bacteria from healthy human volunteers and zoo animals to subsist on antibiotics. Nine gut isolates of Escherichia coli and Cellulosimicrobium sp. displayed increases in colony forming units (CFU) during incubations in minimal medium with only antibiotics added, i.e., the antibiotic subsistence phenotype. Furthermore, laboratory strains of E. coli and Pseudomonas putida equipped with the aminoglycoside 3′ phosphotransferase II gene also displayed the subsistence phenotype on aminoglycosides. In order to address which endogenous genes could be involved in these subsistence phenotypes, the broad-range glycosyl-hydrolase inhibiting iminosugar deoxynojirimycin (DNJ) was used. Addition of DNJ to minimal medium containing glucose showed initial growth retardation of resistant E. coli, which was rapidly recovered to normal growth. In contrast, addition of DNJ to minimal medium containing kanamycin arrested resistant E. coli growth, suggesting that glycosyl-hydrolases were involved in the subsistence phenotype. However, antibiotic degradation experiments showed no reduction in kanamycin, even though the number of CFUs increased. Although antibiotic subsistence phenotypes are readily observed in bacterial species, and are even found in susceptible laboratory strains carrying standard resistance genes, we conclude there is a discrepancy between the observed antibiotic subsistence phenotype and actual antibiotic degradation. Based on these results we can hypothesize that aminoglycoside modifying enzymes might first inactivate the antibiotic (i.e., by acetylation of amino groups, modification of hydroxyl groups by adenylation and phosphorylation respectively), before the subsequent action of catabolic enzymes. Even though we do not dispute that antibiotics could be used as a single carbon source, our observations

  3. Induction of vascular endothelial phenotype and cellular proliferation from human cord blood stem cells cultured in simulated microgravity

    NASA Astrophysics Data System (ADS)

    Chiu, Brian; Z-M Wan, Jim; Abley, Doris; Akabutu, John

    2005-05-01

    Recent studies have demonstrated that stem cells derived from adult hematopoietic tissues are capable of trans-differentiation into non-hematopoietic cells, and that the culture in microgravity ( μg) may modulate the proliferation and differentiation. We investigated the application of μg to human umbilical cord blood stem cells (CBSC) in the induction of vascular endothelial phenotype expression and cellular proliferation. CD34+ mononuclear cells were isolated from waste human umbilical cord blood samples and cultured in simulated μg for 14 days. The cells were seeded in rotary wall vessels (RWV) with or without microcarrier beads (MCB) and vascular endothelial growth factor was added during culture. Controls consisted of culture in 1 G. The cell cultures in RWV were examined by inverted microscopy. Cell counts, endothelial cell and leukocyte markers performed by flow-cytometry and FACS scan were assayed at days 1, 4, 7 and at the termination of the experiments. Culture in RWV revealed significantly increased cellular proliferation with three-dimensional (3D) tissue-like aggregates. At day 4, CD34+ cells cultured in RWV bioreactor without MCB developed vascular tubular assemblies and exhibited endothelial phenotypic markers. These data suggest that CD34+ human umbilical cord blood progenitors are capable of trans-differentiation into vascular endothelial cell phenotype and assemble into 3D tissue structures. Culture of CBSC in simulated μg may be potentially beneficial in the fields of stem cell biology and somatic cell therapy.

  4. Evolutionary Conservation and Network Structure Characterize Genes of Phenotypic Relevance for Mitosis in Human

    PubMed Central

    del Sol, Antonio

    2012-01-01

    The impact of gene silencing on cellular phenotypes is difficult to establish due to the complexity of interactions in the associated biological processes and pathways. A recent genome-wide RNA knock-down study both identified and phenotypically characterized a set of important genes for the cell cycle in HeLa cells. Here, we combine a molecular interaction network analysis, based on physical and functional protein interactions, in conjunction with evolutionary information, to elucidate the common biological and topological properties of these key genes. Our results show that these genes tend to be conserved with their corresponding protein interactions across several species and are key constituents of the evolutionary conserved molecular interaction network. Moreover, a group of bistable network motifs is found to be conserved within this network, which are likely to influence the network stability and therefore the robustness of cellular functioning. They form a cluster, which displays functional homogeneity and is significantly enriched in genes phenotypically relevant for mitosis. Additional results reveal a relationship between specific cellular processes and the phenotypic outcomes induced by gene silencing. This study introduces new ideas regarding the relationship between genotype and phenotype in the context of the cell cycle. We show that the analysis of molecular interaction networks can result in the identification of genes relevant to cellular processes, which is a promising avenue for future research. PMID:22577488

  5. The PLIN4 variant rs8887 modulates obesity related phenotypes in humans through creation of a novel miR-522 seed site

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PLIN4 is a member of the PAT family of lipid storage droplet (LSD) proteins. Associations between seven single nucleotide polymorphisms (SNPs) at human PLIN4 with obesity related phenotypes were investigated using meta-analysis followed by a determination if these phenotypes are modulated by intera...

  6. Individual Human Metabolic Phenotype Analyzed by (1)H NMR of Saliva Samples.

    PubMed

    Wallner-Liebmann, Sandra; Tenori, Leonardo; Mazzoleni, Antonio; Dieber-Rotheneder, Martina; Konrad, Manuela; Hofmann, Peter; Luchinat, Claudio; Turano, Paola; Zatloukal, Kurt

    2016-06-01

    Saliva is an important physiological fluid that contains a complex mixture of analytes that may produce a characteristic individual signature. In recent years, it has been demonstrated that urine possesses a clear signature of the individual metabolic phenotype. Here NMR-based metabolomics was employed to analyze saliva from 23 healthy volunteers. About six saliva samples were collected daily from each individual for 10 consecutive days: 7 days in a real-life situation (days 1-7, Phase I) and 3 days (days 8-10, Phase II) under a standardized diet plus a physical exercise program at day 10. The result is the first demonstration of the existence of an individual metabolic phenotype in saliva. A systematic comparative analysis with urine samples from the same collection scheme demonstrates that the individual phenotype in saliva is slightly weaker than that in urine but less influenced by diet. PMID:27087681

  7. Phenotypic and genotypic association of epithelial IL1RL1 to human TH2-like asthma

    PubMed Central

    Traister, Russell S.; Uvalle, Crystal E.; Hawkins, Gregory A.; Meyers, Deborah A.; Bleecker, Eugene R.; Wenzel, Sally E.

    2014-01-01

    Background Severe asthma remains poorly characterized, although it likely consists of at least 1 phenotype with features of TH2-like inflammation. IL1RL1, encoding both the IL-33 receptor, ST2L, and decoy receptor, sST2, has been genetically associated with asthma, though the mechanism for susceptibility remains unknown. Objective Given previous data supporting a role for IL1RL1 in TH2 inflammation, we hypothesized that ST2L expression might be increased in TH2-like asthma and that expression levels would be associated with single nucleotide polymorphisms in IL1RL1, possibly explaining its genetic relationship with asthma. We also sought to evaluate the regulation of ST2L and sST2 in vitro. Methods Endobronchial brushings and biopsies were obtained and expression of ST2L compared by severity levels, as well as by TH2-like biomarkers. Subjects were genotyped and the relationship of dichotomous expression of ST2L and sST2 to single nucleotide polymorphisms in IL1RL1 were determined. Epithelial cells were grown in air-liquid interface culture, and ST2L and sST2 responses to IFN-γ and IL-13 were evaluated. Results ST2L expression was increased in severe asthma (P = .02) and associated with multiple indicators of TH2-like inflammation, including blood eosinophils (P = .001), exhaled nitric oxide (P = .003), and epithelial CLCA1 (P < .0001) and eotaxin-3 (P = .001) mRNA expression. Multiple single nucleotide polymorphisms in IL1RL1 were found in relation to dichotomous expression of both ST2L and sST2. sST2 expression was associated with IFN-γ expression in bronchoalveolar lavage, while inducing its expression in vitro in primary human epithelial cells. Conclusion Both pathologic and genetic approaches support a role for IL1RL1 in severe asthma, as well as TH2-lke asthma, suggesting that targeting this pathway may have therapeutic benefits. PMID:25091434

  8. Glucocorticoids promote structural and functional maturation of foetal cardiomyocytes: a role for PGC-1α

    PubMed Central

    Rog-Zielinska, E A; Craig, M-A; Manning, J R; Richardson, R V; Gowans, G J; Dunbar, D R; Gharbi, K; Kenyon, C J; Holmes, M C; Hardie, D G; Smith, G L; Chapman, K E

    2015-01-01

    Glucocorticoid levels rise dramatically in late gestation to mature foetal organs in readiness for postnatal life. Immature heart function may compromise survival. Cardiomyocyte glucocorticoid receptor (GR) is required for the structural and functional maturation of the foetal heart in vivo, yet the molecular mechanisms are largely unknown. Here we asked if GR activation in foetal cardiomyocytes in vitro elicits similar maturational changes. We show that physiologically relevant glucocorticoid levels improve contractility of primary-mouse-foetal cardiomyocytes, promote Z-disc assembly and the appearance of mature myofibrils, and increase mitochondrial activity. Genes induced in vitro mimic those induced in vivo and include PGC-1α, a critical regulator of cardiac mitochondrial capacity. SiRNA-mediated abrogation of the glucocorticoid induction of PGC-1α in vitro abolished the effect of glucocorticoid on myofibril structure and mitochondrial oxygen consumption. Using RNA sequencing we identified a number of transcriptional regulators, including PGC-1α, induced as primary targets of GR in foetal cardiomyocytes. These data demonstrate that PGC-1α is a key mediator of glucocorticoid-induced maturation of foetal cardiomyocyte structure and identify other candidate transcriptional regulators that may play critical roles in the transition of the foetal to neonatal heart. PMID:25361084

  9. Approximate distribution of dose among foetal organs for radioiodine uptake via placenta transfer

    NASA Astrophysics Data System (ADS)

    Millard, R. K.; Saunders, M.; Palmer, A. M.; Preece, A. W.

    2001-11-01

    Absorbed radiation doses to internal foetal organs were calculated according to the medical internal radiation dose (MIRD) technique in this study. Anthropomorphic phantoms of the pregnant female as in MIRDOSE3 enabled estimation of absorbed dose to the whole foetus at two stages of gestation. Some foetal organ self-doses could have been estimated by invoking simple spherical models for thyroid, liver, etc, but we investigated the use of the MIRDOSE3 new-born phantom as a surrogate for the stage 3 foetus, scaled to be compatible with total foetal body mean absorbed dose/cumulated activity. We illustrate the method for obtaining approximate dose distribution in the foetus near term following intake of 1 MBq of 123I, 124I, 125I or 131I as sodium iodide by the mother using in vivo biodistribution data examples from a good model of placenta transfer. Doses to the foetal thyroid of up to 1.85 Gy MBq-1 were predicted from the 131I uptake data. Activity in the foetal thyroid was the largest contributor to absorbed dose in the foetal body, brain, heart and thymus. Average total doses to the whole foetus ranged from 0.16 to 1.2 mGy MBq-1 for stages 1 and 3 of pregnancy using the MIRDOSE3 program, and were considerably higher than those predicted from the maternal contributions alone. Doses to the foetal thymus and stomach were similar, around 2-3 mGy MBq-1. Some foetal organ doses from the radioiodides were ten times higher than to the corresponding organs of the mother, and up to 100 times higher to the thyroid. The fraction of activity uptakes in foetal organs were distributed similarly to the maternal ones.

  10. Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters

    PubMed Central

    Bröer, Stefan; Bailey, Charles G.; Kowalczuk, Sonja; Ng, Cynthia; Vanslambrouck, Jessica M.; Rodgers, Helen; Auray-Blais, Christiane; Cavanaugh, Juleen A.; Bröer, Angelika; Rasko, John E.J.

    2008-01-01

    Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes. PMID:19033659

  11. MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure.

    PubMed

    Kosten, Ilona J; Spiekstra, Sander W; de Gruijl, Tanja D; Gibbs, Susan

    2015-08-15

    After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a(+) MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a(-)/CD14(+)/CD68(+) which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets. PMID:26028481

  12. Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

    SciTech Connect

    Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

    1987-12-01

    The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2/sup +/, CD3/sup +/, CD4/sup +/ or CD2/sup +/, CD3/sup +/, CD8/sup +/) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by /sup 51/Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype.

  13. Activation of endogenous human stem cell-associated retroviruses (SCARs) and therapy-resistant phenotypes of malignant tumors.

    PubMed

    Glinsky, Gennadi V

    2016-07-01

    Recent reports revealed consistent activation of specific endogenous retroviral elements in human preimplantation embryos and embryonic stem cells. Activity of stem cell associated retroviruses (SCARs) has been implicated in seeding thousands of human-specific regulatory sequences in the hESC genome. Activation of specific SCARs has been demonstrated in patients diagnosed with multiple types of cancer, autoimmune diseases, and neurodegenerative disorders, and appears associated with clinically lethal therapy resistant death-from-cancer phenotypes in a sub-set of cancer patients diagnosed with different types of malignant tumors. A hallmark feature of human-specific SCAR integration sites is deletions of ancestral DNA. Analysis of human-specific genetic loci of SCARs' stemness networks in tumor samples of TCGA cohorts representing 29 cancer types suggests that this approach may facilitate identification of pan-cancer genomic signatures of clinically-lethal disease defined by the presence of somatic non-silent mutations, gene-level copy number changes, and transcripts and proteins' expression of SCAR-regulated host genes. Present analyses indicate that multiple lines of strong circumstantial evidence support the hypothesis that activation of SCARs' networks may play an important role in cancer progression and metastasis, perhaps contributing to the emergence of clinically-lethal therapy-resistant death-from-cancer phenotypes. PMID:27084523

  14. Two-dimensional culture of human pancreatic adenocarcinoma cells results in an irreversible transition from epithelial to mesenchymal phenotype

    PubMed Central

    Kang, Ya'an; Zhang, Ran; Suzuki, Rei; Li, Shao-qiang; Roife, David; Truty, Mark J.; Chatterjee, Deyali; Thomas, Ryan M.; Cardwell, James; Wang, Yu; Wang, Huamin; Katz, Matthew H.; Fleming, Jason B.

    2015-01-01

    Many commercially available cell lines have been in culture for ages, acquiring phenotypes that differ from the original cancers from which these cell lines were derived. Therefore, research on new cell lines could improve the success rates of translational research in cancer. We have developed methods for the isolation and culture of human pancreatic ductal adenocarcinoma (PDAC) cells from murine xenografts of human PDAC. We hypothesize that phenotypes of PDAC cells are modified by in vitro culture conditions over time and by in vivo implantation. Patient-derived xenografts were created in immunodeficient mice using surgically resected tumor specimens. These murine xenografts were then used to establish human PDAC cell lines in culture. Earlier (<5) passage and later (>20) passage cell lines were evaluated separately regarding proliferation, cell cycle, genetic mutations, invasiveness, chemosensitivity, tumorigenesis, epithelial-mesenchymal transition (EMT) status, and proteomics. Later passage cells accelerated their doubling time and colony formation, and were more concentrated in the G0/G1 phase and less in the G2/M checkpoint phase. Later passage cells were more sensitive to gemcitabine and 5-fluorouracil than earlier passage cells, but all four new cell lines were more chemo-resistant compared to commercial ATCC cell lines. EMT induction was observed when establishing and passaging cell lines in vitro and furthermore by growing them as subcutaneous tumors in vivo. This study demonstrates a novel approach to the establishment of PDAC cell lines and observes a process by which newly established cell lines undergo phenotypic changes during in vitro culture and in vivo tumorigenesis. This may help explain differences of treatment effects often observed between experiments conducted in vitro, in vivo, and in human clinical trials. PMID:25485535

  15. Pathophysiology of foetal oxygenation and cell damage during labour.

    PubMed

    Yli, Branka M; Kjellmer, Ingemar

    2016-01-01

    A foetus exposed to oxygenation compromise is capable of several adaptive responses, which can be categorised into those affecting metabolism and those affecting oxygen transport. However, both the extent and duration of the impairment in oxygenation will have a bearing on these adaptive responses. Although intrapartum events may account for no more than one-third of cases with an adverse neurological outcome, they are important because they can be influenced successfully. This review describes the mechanisms underlying foetal hypoxia during labour, acid-base balance and gas exchange, and the current scientific understanding of the role of intrauterine asphyxia in the pathophysiology of neonatal encephalopathy and cerebral palsy. Although the mechanisms involved include similar initiating events, principally ischaemia and excitotoxicity, and similar final common pathways to cell death, there are certain unique maturational factors that influence the type and pattern of cellular injury. PMID:26211833

  16. Comparing methods for ex vivo characterization of human monocyte phenotypes and in vitro responses.

    PubMed

    Johnston, Lisa; Harding, Scott A; La Flamme, Anne Camille

    2015-12-01

    Monocytes are key innate effector cells and their phenotype and function may be a useful biomarker of disease state or therapeutic response. However, for such an assay to be clinically feasible it needs to be simple and reproducible, which this study aimed to address. Peripheral blood mononuclear cells (PBMC)(2) isolated from whole blood using Histopaque-1077 or cell preparation tubes (CPT) showed no difference in the ex vivo monocyte activation marker expression or in vitro responses; however, a delayed isolation using CPT significantly altered ex vivo and in vitro phenotypes and responses. Furthermore, purification of monocytes using CD14(+) microbeads resulted in a loss of CD14(low)CD16(+) monocytes compared to PBMC samples. Thus, the use of CPT reduced complexity and time compared to Histopaque, and PBMC isolation allowed the analysis of all 3 major monocyte subsets. Finally, because the delayed isolation of PBMC from CPT significantly altered monocytes, time delays should be standardized. PMID:26256247

  17. What Can hiPSC-Cardiomyocytes Teach Us about Modeling Complex Human Disease Phenotypes?

    PubMed

    Broeckel, Ulrich

    2016-09-01

    Using induced pluripotent stem cell-derived cardiomyocytes to predict patient-specific responses to drug treatments requires that they maintain inter-individual variability for RNA expression and recapitulate phenotypic responses in vitro. In this issue of Cell Stem Cell, Matsa et al. (2016) show that expression differences between hiPSC-cardiomyocytes provide a personalized drug testing platform to evaluate potential efficacy and cardiotoxicity. PMID:27588742

  18. Xenopus pax6 mutants affect eye development and other organ systems, and have phenotypic similarities to human aniridia patients.

    PubMed

    Nakayama, Takuya; Fisher, Marilyn; Nakajima, Keisuke; Odeleye, Akinleye O; Zimmerman, Keith B; Fish, Margaret B; Yaoita, Yoshio; Chojnowski, Jena L; Lauderdale, James D; Netland, Peter A; Grainger, Robert M

    2015-12-15

    Mutations in the Pax6 gene cause ocular defects in both vertebrate and invertebrate animal species, and the disease aniridia in humans. Despite extensive experimentation on this gene in multiple species, including humans, we still do not understand the earliest effects on development mediated by this gene. This prompted us to develop pax6 mutant lines in Xenopus tropicalis taking advantage of the utility of the Xenopus system for examining early development and in addition to establish a model for studying the human disease aniridia in an accessible lower vertebrate. We have generated mutants in pax6 by using Transcription Activator-Like Effector Nuclease (TALEN) constructs for gene editing in X. tropicalis. Embryos with putative null mutations show severe eye abnormalities and changes in brain development, as assessed by changes in morphology and gene expression. One gene that we found is downregulated very early in development in these pax6 mutants is myc, a gene involved in pluripotency and progenitor cell maintenance and likely a mediator of some key pax6 functions in the embryo. Changes in gene expression in the developing brain and pancreas reflect other important functions of pax6 during development. In mutations with partial loss of pax6 function eye development is initially relatively normal but froglets show an underdeveloped iris, similar to the classic phenotype (aniridia) seen in human patients with PAX6 mutations. Other eye abnormalities observed in these froglets, including cataracts and corneal defects, are also common in human aniridia. The frog model thus allows us to examine the earliest deficits in eye formation as a result of pax6 lesions, and provides a useful model for understanding the developmental basis for the aniridia phenotype seen in humans. PMID:25724657

  19. Functional and phenotypic characteristics of alternative activation induced in human monocytes by interleukin-4 or the parasitic nematode Brugia malayi.

    PubMed

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Moore, Vanessa; Sanprasert, Vivornpun; Nutman, Thomas B

    2011-10-01

    Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly. PMID:21788379

  20. Non-ABO blood group systems phenotyping in non-human primates for blood banking laboratory and xenotransplantation.

    PubMed

    Ramis, G; Martínez-Alarcon, L; Quereda, J J; Mrowiec, A; Funes, C; Ríos, A; Ramírez, P; Muñoz, A; Majado, M J

    2013-04-01

    Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion. PMID:23563364

  1. Mapping quantitative-trait loci in humans by use of extreme concordant sib pairs: Selected sampling by parental phenotypes

    SciTech Connect

    Zhang, Heping; Risch, N.

    1996-10-01

    In two previous articles, we have considered sample sizes required to detect linkage for mapping quantitative-trait loci in humans, using extreme discordant sib pairs. Here, we examine further the use of extreme concordant sib pairs but consider the effect of parents` phenotypes. Sample sizes necessary to obtain a power of 80% with concordant sib pairs at a significance level of .0001 are given, stratified by parental phenotypes. When there is no residual correlation between sibs, the parental phenotypes have little impact on the sample sizes. When residual correlations between sibs exist, we show, however, that power can be considerably reduced by including extreme sib pairs when the parents also have similarly extreme values. Thus, we recommend the exclusion of such pairs from linkage studies. This recommendation reduces the required sample sizes by 3- to 28-fold. The degree of saving in the required sample sizes varies among different models and allele frequencies. The reduction is most dramatic (a 28-fold reduction) for a rare recessive gene. 5 refs., 6 tabs.

  2. Recombinant virus assay: a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates.

    PubMed Central

    Kellam, P; Larder, B A

    1994-01-01

    Antiviral drug susceptibility assays for clinical human immunodeficiency virus type 1 (HIV-1) isolates are required to monitor the development of drug resistance during clinical trials and antiretroviral drug therapy. First-generation phenotypic assays possess a number of drawbacks, not least the selection of unrepresentative virus populations during cocultivation. Here we describe a rapid phenotypic assay for the assessment of the susceptibility of clinical isolates to reverse transcriptase (RT) inhibitors. This procedure, called the recombinant virus assay, allows the generation of viable virus by homologous recombination of a PCR-derived pool of RT coding sequences into an RT-deleted, noninfectious proviral clone, pHIV delta BstEII. A nested PCR procedure has been optimized to allow the amplification of an RT pool from both uncultured and cocultured infected patient peripheral blood lymphocyte (PBL) DNA for subsequent use in the creation of recombinant viruses. Analysis of two patients during the course of zidovudine therapy showed that this approach produced viruses which accurately exhibited the same genotype and phenotype as that of the original infected PBL DNA. The recombinant virus assay can be performed in approximately 3 weeks without the use of donor PBLs and therefore represents a rapid, nonselective procedure for the assay of clinical isolates. Images PMID:8141575

  3. Non-invasive Foetal ECG – a Comparable Alternative to the Doppler CTG?

    PubMed Central

    Reinhard, J.; Louwen, F.

    2012-01-01

    This review discusses the alternative of using the non-invasive foetal ECG compared with the conventionally used Doppler CTG. Non-invasive abdominal electrocardiograms (ECG) have been approved for clinical routine since 2008; subsequently they were also approved for antepartum and subpartum procedures. The first study results have been published. Non-invasive foetal ECG is especially indicated during early pregnancy, while the Doppler CTG is recommended for the vernix period. Beyond the vernix period no difference has been recorded in the success rate of either approach. The foetal ECG signal quality is independent of the BMI, whereas the success rate of the Doppler CTG is diminished with an increased BMI. During the first stage of labour, non-invasive foetal ECG demonstrates better signal quality; however during the second stage of labour no difference has been identified between the methods. PMID:25308981

  4. PEG3 domain gene expression in maternal and foetal placenta in intrauterine growth restricted bovine foetuses.

    PubMed

    Li, Shun; Pausch, Hubert; Venhoranta, Heli; Adamowicz, Krzysztof; Andersson, Magnus; Zwierzchowski, Lech; Kind, Alexander; Schnieke, Angelika; Flisikowski, Krzysztof

    2016-02-01

    We used a genetic (MIMT1(Del)) model of intrauterine growth restriction to investigate dysregulation of PEG3 domain gene expression in bovine foetal and maternal placenta. ZIM2, APEG3 and PEG3 expressions were similarly reduced in MIMT1(Del/) (WT) foetal placenta, suggesting coordinated regulation. Methylation of DNA CpG sites associated with these genes showed no differences, but differences in the levels of MIMT1 RNA methylation at three CpG sites were found in foetal placenta. Our data are consistent with the presence of a bidirectional promoter 5' of MIMT1 and suggest a regulatory role for the MIMT1 non-coding transcript. PEG3 domain expression on the maternal placenta side was not affected by the foetal mutation. PMID:26537866

  5. Phthalate-Induced Pathology in the Foetal Testis Involves More Than Decreased Testosterone Production

    EPA Science Inventory

    Foetal exposure to phthalates is known to adversely impact male reproductive development and function. Developmental anomalies of reproductive tract have been attributed to impaired testosterone synthesis. However, species differences in the ability to produce testosterone have...

  6. Notochordal conditioned media from tissue increases proteoglycan accumulation and promotes a healthy nucleus pulposus phenotype in human mesenchymal stem cells

    PubMed Central

    2011-01-01

    Introduction Notochordal cells (NCs) are influential in development of the intervertebral disc (IVD) and species that retain NCs do not degenerate. IVD repair using bone marrow derived mesenchymal stem cells (MSCs) is an attractive approach and the harsh microenvironment of the IVD suggests pre-differentiation is a necessary first step. The goal of this study was to use soluble factors from NCs in alginate and NCs in their native tissue to differentiate human MSCs to a young nucleus pulposus (NP) phenotype. Methods Human MSCs (cultured under micromass conditions for 21 days in hypoxia) were differentiated with conditioned medium derived from porcine notochordal cells in native tissue (NCT) or in alginate beads (NCA), and compared with chondrogenic (TGFβ-3) or basal medium. A PCR array of 42 genes was utilized to screen a large number of genes known to be associated with the healthy NP phenotype and pellet cultures were also evaluated for glycosaminoglycan content, histology and viability. Proteomic analysis was used to assess candidate soluble factors in NCA and NCT. Results Notochordal cell conditioned media had diverse effects on MSC phenotype. NCT resulted in the highest levels of glycosaminoglycan (GAG), as well as up-regulation of SOX9 and Collagen II gene expression. NCA demonstrated effects that were catabolic yet also anti-fibrotic and minimally hypertrophic with down-regulation of Collagens I and III and low levels of Collagen X, respectively. Micromass culture and hypoxic conditions were sufficient to promote chondrogenesis demonstrating that both basal and chondrogenic media produced similar phenotypes. Candidate matricellular proteins, clusterin and tenascin were identified by proteomics in the NCA group. Conclusions NCs secreted important soluble factors capable of differentiating MSCs to a NP phenotype synthesizing high levels of proteoglycan while also resisting collagen fiber expression and hypertrophy, yet results were sensitive to the conditions

  7. Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact.

    PubMed

    Fornara, O; Bartek, J; Rahbar, A; Odeberg, J; Khan, Z; Peredo, I; Hamerlik, P; Bartek, J; Stragliotto, G; Landázuri, N; Söderberg-Nauclér, C

    2016-02-01

    Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor. PMID:26138445

  8. A systematic analysis of recombination activity and genotype-phenotype correlation in human recombination-activating gene 1 deficiency

    PubMed Central

    Lee, Yu Nee; Frugoni, Francesco; Dobbs, Kerry; Walter, Jolan E.; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Haddad, Elie; LeDeist, Francoise; Bleesing, Jack H.; Henderson, Lauren A.; Pai, Sung-Yun; Nelson, Robert P.; El-Ghoneimy, Dalia H.; El-Feky, Reem A.; Reda, Shereen M.; Hossny, Elham; Soler-Palacin, Pere; Fuleihan, Ramsay L.; Patel, Niraj C.; Massaad, Michel J.; Geha, Raif S.; Puck, Jennifer M.; Palma, Paolo; Cancrini, Caterina; Chen, Karin; Vihinen, Mauno; Alt, Frederick W.; Notarangelo, Luigi D.

    2014-01-01

    Background The recombination-activating gene (RAG) 1/2 proteins play a critical role in the development of T and B cells by initiating the VDJ recombination process that leads to generation of a broad T-cell receptor (TCR) and B-cell receptor repertoire. Pathogenic mutations in the RAG1/2 genes result in various forms of primary immunodeficiency, ranging from T−B− severe combined immune deficiency to delayed-onset disease with granuloma formation, autoimmunity, or both. It is not clear what contributes to such heterogeneity of phenotypes. Objective We sought to investigate the molecular basis for phenotypic diversity presented in patients with various RAG1 mutations. Methods We have developed a flow cytometry–based assay that allows analysis of RAG recombination activity based on green fluorescent protein expression and have assessed the induction of the Ighc locus rearrangements in mouse Rag1−/− pro-B cells reconstituted with wild-type or mutant human RAG1 (hRAG1) using deep sequencing technology. Results Here we demonstrate correlation between defective recombination activity of hRAG1 mutant proteins and severity of the clinical and immunologic phenotype and provide insights on the molecular mechanisms accounting for such phenotypic diversity. Conclusions Using a sensitive assay to measure the RAG1 activity level of 79 mutations in a physiologic setting, we demonstrate correlation between recombination activity of RAG1 mutants and the severity of clinical presentation and show that RAG1 mutants can induce specific abnormalities of the VDJ recombination process. PMID:24290284

  9. Introduction: Why is intrapartum foetal monitoring necessary - Impact on outcomes and interventions.

    PubMed

    Ayres-de-Campos, Diogo

    2016-01-01

    Maintaining maternal oxygen supply is essential for foetal life, and labour constitutes an increased challenge to this. Good clinical judgement is required to evaluate the signs of reduced foetal oxygenation, to diagnose the underlying cause, to judge the reversibility of the condition and to determine the best timing for delivery. The main aim of intrapartum foetal monitoring is to identify foetuses that are being inadequately oxygenated, enabling appropriate action before the occurrence of injury. It is also to provide reassurance in cases of adequate foetal oxygenation, and thus to avoid unnecessary obstetric intervention. Poor foetal oxygenation is diagnosed by documenting metabolic acidosis in the umbilical cord immediately after birth or in the newborn circulation during the first minutes of life. However, most newborns recover quickly, and they do not develop relevant short- or long-term complications. Hypoxic-ischaemic encephalopathy is the short-term neurological dysfunction caused by inadequate intrapartum foetal oxygenation, and cerebral palsy of the spastic quadriplegic or dyskinetic types is the long-term neurological complication most commonly associated with it. Although there is insufficient evidence from randomised controlled trials to demonstrate that any form of intrapartum foetal monitoring reduces the incidence of adverse outcomes, reports from the clinical setting have documented a decrease in metabolic acidosis, hypoxic-ischaemic encephalopathy and intrapartum death over the last decades. It may be difficult to demonstrate the benefit of diagnostic techniques in complex environments such as the labour ward, but a reduction in the incidence of adverse clinical outcomes constitutes important evidence that intrapartum foetal monitoring makes a difference. PMID:26189688

  10. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

    SciTech Connect

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J.

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  11. An ECG simulator for generating maternal-foetal activity mixtures on abdominal ECG recordings.

    PubMed

    Behar, Joachim; Andreotti, Fernando; Zaunseder, Sebastian; Li, Qiao; Oster, Julien; Clifford, Gari D

    2014-08-01

    Accurate foetal electrocardiogram (FECG) morphology extraction from non-invasive sensors remains an open problem. This is partly due to the paucity of available public databases. Even when gold standard information (i.e derived from the scalp electrode) is present, the collection of FECG can be problematic, particularly during stressful or clinically important events.In order to address this problem we have introduced an FECG simulator based on earlier work on foetal and adult ECG modelling. The open source foetal ECG synthetic simulator, fecgsyn, is able to generate maternal-foetal ECG mixtures with realistic amplitudes, morphology, beat-to-beat variability, heart rate changes and noise. Positional (rotation and translation-related) movements in the foetal and maternal heart due to respiration, foetal activity and uterine contractions were also added to the simulator.The simulator was used to generate some of the signals that were part of the 2013 PhysioNet Computing in Cardiology Challenge dataset and has been posted on Physionet.org (together with scripts to generate realistic scenarios) under an open source license. The toolbox enables further research in the field and provides part of a standard for industry and regulatory testing of rare pathological scenarios. PMID:25071094

  12. Phenotypic and genotypic characterization of a new fish-virulent Vibrio vulnificus serovar that lacks potential to infect humans.

    PubMed

    Fouz, Belén; Roig, Francisco J; Amaro, Carmen

    2007-06-01

    Vibrio vulnificus is a bacterial species that is virulent for humans and fish. Human isolates are classified into biotypes 1 and 3 (BT1 and BT3) and fish isolates into biotype 2 (BT2). However, a few human infections caused by BT2 isolates have been reported worldwide (zoonosis). These BT2 human isolates belong to serovar E (SerE), which is also present in diseased fish. The aim of the present work was to characterize a new BT2 serovar [serovar A (SerA)], which emerged in the European fish-farming industry in 2000, by means of phenotypic, serological and genetic [plasmid profiling, ribotyping and random amplified polymorphic DNA (RAPD)] methodologies. The results confirmed that SerA constitutes a homogeneous O-serogroup within the species that shares plasmidic information with SerE. Like SerE, this new serogroup was resistant to fresh fish serum, as well as being highly virulent for fish. In contrast, it was sensitive to human serum and avirulent for mice, even after pretreatment with iron. The two serovars presented different biochemical profiles as well as specific patterns by ribotyping and RAPD analysis. In conclusion, SerA seems to constitute a different clonal group that has recently emerged within the species V. vulnificus, with pathogenic potential for fish but not for humans. PMID:17526849

  13. Thrombospondin 1 promotes an aggressive phenotype through epithelial-to-mesenchymal transition in human melanoma

    PubMed Central

    Jayachandran, Aparna; Anaka, Matthew; Prithviraj, Prashanth; Hudson, Christopher; McKeown, Sonja J; Lo, Pu-Han; Vella, Laura J; Goding, Colin R; Cebon, Jonathan; Behren, Andreas

    2014-01-01

    Epithelial-to-mesenchymal transition (EMT), in which epithelial cells loose their polarity and become motile mesenchymal cells, is a determinant of melanoma metastasis. We compared gene expression signatures of mesenchymal-like melanoma cells with those of epithelial-like melanoma cells, and identified Thrombospondin 1 (THBS1) as highly up-regulated in the mesenchymal phenotype. This study investigated whether THBS1, a major physiological activator of transforming growth factor (TGF)-beta, is involved in melanoma EMT-like process. We sought to examine expression patterns in distinct melanoma phenotypes including invasive, de-differentiated, label-retaining and drug resistant populations that are putatively associated with an EMT-like process. Here we show that THBS1 expression and secretion was elevated in melanoma cells exhibiting invasive, drug resistant, label retaining and mesenchymal phenotypes and correlated with reduced expression of genes involved in pigmentation. Elevated THBS1 levels were detected in Vemurafenib resistant melanoma cells and inhibition of THBS1 led to significantly reduced chemoresistance in melanoma cells. Notably, siRNA-mediated silencing of THBS1 and neutralizing antibody to THBS1 reduced invasion in mesenchymal-like melanoma cells, while ectopic THBS1 expression in epithelial-like melanoma cells enhanced invasion. Furthermore, the loss of THBS1 inhibited in vivo motility of melanoma cells within the embryonic chicken neural tube. In addition, we found aberrant THBS1 protein expression in metastatic melanoma tumor biopsies. These results implicate a role for THBS1 in EMT, and hence THBS1 may serve as a novel target for strategies aimed at the treatment of melanoma invasion and drug resistance. PMID:25051363

  14. Efficient in vitro RNA interference and immunofluorescence-based phenotype analysis in a human parasitic nematode, Brugia malayi

    PubMed Central

    2012-01-01

    Background RNA interference (RNAi) is an efficient reverse genetics technique for investigating gene function in eukaryotes. The method has been widely used in model organisms, such as the free-living nematode Caenorhabditis elegans, where it has been deployed in genome-wide high throughput screens to identify genes involved in many cellular and developmental processes. However, RNAi techniques have not translated efficiently to animal parasitic nematodes that afflict humans, livestock and companion animals across the globe, creating a dependency on data tentatively inferred from C. elegans. Results We report improved and effective in vitro RNAi procedures we have developed using heterogeneous short interfering RNA (hsiRNA) mixtures that when coupled with optimized immunostaining techniques yield detailed analysis of cytological defects in the human parasitic nematode, Brugia malayi. The cellular disorganization observed in B. malayi embryos following RNAi targeting the genes encoding γ-tubulin, and the polarity determinant protein, PAR-1, faithfully phenocopy the known defects associated with gene silencing of their C. elegans orthologs. Targeting the B. malayi cell junction protein, AJM-1 gave a similar but more severe phenotype than that observed in C. elegans. Cellular phenotypes induced by our in vitro RNAi procedure can be observed by immunofluorescence in as little as one week. Conclusions We observed cytological defects following RNAi targeting all seven B. malayi transcripts tested and the phenotypes mirror those documented for orthologous genes in the model organism C. elegans. This highlights the reliability, effectiveness and specificity of our RNAi and immunostaining procedures. We anticipate that these techniques will be widely applicable to other important animal parasitic nematodes, which have hitherto been mostly refractory to such genetic analysis. PMID:22243803

  15. Heparin-based self-assembling peptide scaffold reestablish chondrogenic phenotype of expanded de-differentiated human chondrocytes.

    PubMed

    Recha-Sancho, Lourdes; Semino, Carlos E

    2016-07-01

    The use of chondrocytes in cell-based therapies for cartilage lesions are limited by quantity and, therefore, require an in vitro expansion. As monolayer culture leads to de-differentiation, different culture techniques are currently under development to recover chondrocyte phenotype after cell expansion. In the present work, we studied the capacity of the bimolecular heparin-based self-assembling peptide scaffold (RAD16-I) as a three-dimensional (3D) culture system to foster reestablishment of chondrogenic phenotype of de-differentiated human Articular Chondrocytes (AC). The culture was performed in a serum-free medium under control and chondrogenic induction and good viability results were observed after 4 weeks of culture in both conditions. Cells changed their morphology to a more elongated shape and established a cellular network that induced the condensation of the constructs in the case of chondrogenic medium, leading to a compacted structure with improved mechanical properties. Specific extracellular matrix (ECM) proteins of mature cartilage, such as collagen type II and aggrecan were up-regulated under chondrogenic medium and significantly enhanced with the presence of heparin in the scaffold. 3D constructs became highly stained with toluidine blue dye after 4 weeks of culture, indicating the presence of synthetized proteoglycans (PGs) by the cells. Interestingly, the full viscoelastic behavior was closely related to that found in chicken native cartilage. Altogether, the results suggest that the 3D culture model described can help de-differentiated human chondrocytes to recover its cartilage phenotype. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1694-1706, 2016. PMID:26939919

  16. Germ cell specific overactivation of WNT/βcatenin signalling has no effect on folliculogenesis but causes fertility defects due to abnormal foetal development

    PubMed Central

    Kumar, Manish; Camlin, Nicole J.; Holt, Janet E.; Teixeira, Jose M.; McLaughlin, Eileen A.; Tanwar, Pradeep S.

    2016-01-01

    All the major components of the WNT signalling pathway are expressed in female germ cells and embryos. However, their functional relevance in oocyte biology is currently unclear. We examined ovaries collected from TCFGFP mice, a well-known Wnt reporter mouse model, and found dynamic changes in the Wnt/βcatenin signalling activity during different stages of oocyte development and maturation. To understand the functional importance of Wnt signalling in oocytes, we developed a mouse model with the germ cell-specific constitutive activation of βcatenin using cre recombinase driven by the DEAD (Asp-Glu-Ala-Asp) box protein 4 (Ddx4) gene promoter. Histopathological and functional analysis of ovaries from these mutant mice (Ctnnb1ex3cko) showed no defects in ovarian functions, oocytes, ovulation and early embryonic development. However, breeding of the Ctnnb1ex3cko female mice with males of known fertility never resulted in birth of mutant pups. Examination of uteri from time pregnant mutant females revealed defects in ectoderm differentiation leading to abnormal foetal development and premature death. Collectively, our work has established the role of active WNT/βcatenin signalling in oocyte biology and foetal development, and provides novel insights into the possible mechanisms of complications in human pregnancy such as repeated spontaneous abortion, sudden intrauterine unexpected foetal death syndrome and stillbirth. PMID:27265527

  17. Germ cell specific overactivation of WNT/βcatenin signalling has no effect on folliculogenesis but causes fertility defects due to abnormal foetal development.

    PubMed

    Kumar, Manish; Camlin, Nicole J; Holt, Janet E; Teixeira, Jose M; McLaughlin, Eileen A; Tanwar, Pradeep S

    2016-01-01

    All the major components of the WNT signalling pathway are expressed in female germ cells and embryos. However, their functional relevance in oocyte biology is currently unclear. We examined ovaries collected from TCFGFP mice, a well-known Wnt reporter mouse model, and found dynamic changes in the Wnt/βcatenin signalling activity during different stages of oocyte development and maturation. To understand the functional importance of Wnt signalling in oocytes, we developed a mouse model with the germ cell-specific constitutive activation of βcatenin using cre recombinase driven by the DEAD (Asp-Glu-Ala-Asp) box protein 4 (Ddx4) gene promoter. Histopathological and functional analysis of ovaries from these mutant mice (Ctnnb1(ex3)cko) showed no defects in ovarian functions, oocytes, ovulation and early embryonic development. However, breeding of the Ctnnb1(ex3)cko female mice with males of known fertility never resulted in birth of mutant pups. Examination of uteri from time pregnant mutant females revealed defects in ectoderm differentiation leading to abnormal foetal development and premature death. Collectively, our work has established the role of active WNT/βcatenin signalling in oocyte biology and foetal development, and provides novel insights into the possible mechanisms of complications in human pregnancy such as repeated spontaneous abortion, sudden intrauterine unexpected foetal death syndrome and stillbirth. PMID:27265527

  18. Cestode Antigens Induce a Tolerogenic-Like Phenotype and Inhibit LPS Inflammatory Responses in Human Dendritic Cells

    PubMed Central

    Terrazas, César A.; Sánchez-Muñoz, Fausto; Mejía-Domínguez, Ana M.; Amezcua-Guerra, Luis M.; Terrazas, Luis I.; Bojalil, Rafael; Gómez-García, Lorena

    2011-01-01

    Pathogens have developed strategies to modify Dendritic Cells (DCs) phenotypes and impair their functions in order to create a safer environment for their survival. DCs responses to helminths and their derivatives vary among different studies. Here we show that excretory/secretory products of the cestode Taenia crassiceps (TcES) do not induce the maturation of human DCs judged by a lack of increment in the expression of CD83, HLA-DR, CD80 and CD86 molecules but enhanced the production of IL-10 and positively modulated the expression of the C-type lectin receptor MGL and negatively modulated the expression of DC-SIGN. Additionally, these antigens were capable of down-modulating the inflammatory response induced by LPS in these cells by reducing the expression of the maturation markers and the production of the inflammatory cytokines IL-1β, TNF, IL-12 and IL-6. The effects of TcES upon the DCs responses to LPS were stronger if cells were exposed during their differentiation to the helminth antigens. All together, these findings suggest the ability of TcES to induce the differentiation of human DCs into a tolerogenic-like phenotype and to inhibit the effects of inflammatory stimuli. PMID:22110390

  19. Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro.

    PubMed

    Roesch-Ely, Mariana; Steinberg, Thorsten; Bosch, F Xavier; Müssig, Eva; Whitaker, Noel; Wiest, Tina; Kohl, Annette; Komposch, Gerda; Tomakidi, Pascal

    2006-12-01

    We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type 16 (HPV16). The approach adopted is based on the OCC system facilitating spatially separated cell growth and cell-to-cell interactions via diffusible growth factors. Generally, IHGK reveal transcription of the HPV16 E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for growth factors interleukin 1beta, granulocyte-macrophage colony stimulating factor, fibroblast growth factor 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant growth. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro. PMID:17177858

  20. Transgenic rats overexpressing the human MrgX3 gene show cataracts and an abnormal skin phenotype

    SciTech Connect

    Kaisho, Yoshihiko . E-mail: Kaisho_Yoshihiko@takeda.co.jp; Watanabe, Takuya; Nakata, Mitsugu; Yano, Takashi; Yasuhara, Yoshitaka; Shimakawa, Kozo; Mori, Ikuo; Sakura, Yasufumi; Terao, Yasuko; Matsui, Hideki; Taketomi, Shigehisa

    2005-05-13

    The human MrgX3 gene, belonging to the mrgs/SNSRs (mass related genes/sensory neuron specific receptors) family, was overexpressed in transgenic rats using the actin promoter. Two animal lines showed cataracts with liquification/degeneration and swelling of the lens fiber cells. The transient epidermal desquamation was observed in line with higher gene expression. Histopathology of the transgenic rats showed acanthosis and focal parakeratosis. In the epidermis, there was an increase in cellular keratin 14, keratin 10, and loricrin, as well as PGP 9.5 in innervating nerve fibers. These phenotypes accompanied an increase in the number of proliferating cells. These results suggest that overexpression of the human MrgX3 gene causes a disturbance of the normal cell-differentiation process.

  1. Ultrastructural and phenotypic characterization of CABA I, a new human ovarian cancer cell line.

    PubMed

    Dolo, V; Ginestra, A; Violini, S; Miotti, S; Festuccia, C; Miceli, D; Migliavacca, M; Rinaudo, C; Romano, F M; Brisdelli, F; Canevari, S; Pavan, A; Vittorelli, M L

    1997-01-01

    We have established an ovarian cancer cell line (CABA I) from ascitic fluid obtained from a patient with papillary adenocarcinoma of the ovary prior to drug treatment. The epithelial origin of the cell line was confirmed by morphology and by immunofluorescence analysis using anticytokeratin antibodies. Ultrastructural analysis revealed a very irregular membrane surface and a clear cytoplasm rich in electron-lucent vesicles. CABA I cells grow rapidly in culture (doubling time 18 h) in an anchorage-independent manner. Exogenously added beta-estradiol and epidermal growth factor (EGF) treatments did not influence cell growth rate. FACS analysis to determine the phenotypic profile of tumor-associated antigen, membrane receptor, and adhesion molecule expression indicated that the cell line was positive for different members of the c-erbB family, for alpha 6 and beta 1 integrin receptors, and intensively positive for HLA class I antigens and the folate receptor. Molecular characterization revealed no mutations for c-myc and c-k-ras genes, but did detect an exon 5 mutation in the p53 gene. CABA I cells grew poorly as heterotransplants in nude mice, and tumors showed long latency periods. Because early (15-20) and late (55-60) passage cells maintain the same growth and phenotypic characteristics, the CABA I cell line might provide a good in vitro model system to investigate the cellular and molecular events involved in ovarian carcinogenesis. PMID:9220498

  2. Adaptive Thermogenesis in Resistance to Obesity Therapies: Issues in Quantifying Thrifty Energy Expenditure Phenotypes in Humans.

    PubMed

    Dulloo, Abdul G; Schutz, Yves

    2015-06-01

    Dieting and exercise are likely to remain the core approaches in the management of obesity in the foreseeable future despite their well-documented failures for achieving long-term weight loss. Explanations for such poor prognosis are centered on patient's self-regulatory failure and lack of compliance to the prescribed diet or exercise regimen. While a role for physiological adaptations leading to diminished rates of heat production has also been advocated, there are considerable uncertainties about the quantitative importance of such regulated heat production (i.e., adaptive thermogenesis) to the less-than-expected weight loss and ease for weight regain. This paper first reviews the most compelling evidence of what is often considered as weight loss-induced adaptive thermogenesis in various compartments of daily energy expenditure. It then discusses the major limitations and issues in quantifying such thrifty energy expenditure phenotypes and underscores the plausibility of diminished core temperature as a thrifty metabolic trait in resistance to weight loss. Although an accurate quantification of adaptive thermogenesis will have to await the applications of deep body composition phenotyping and better discrimination of physical activity energy expenditures, the magnitude of diminished energy expenditure in response to weight loss in certain individuals is large enough to support the concept that adaptive thermogenesis contribute importantly to their resistance to obesity therapies. PMID:26627218

  3. EGF shifts human airway basal cell fate toward a smoking-associated airway epithelial phenotype.

    PubMed

    Shaykhiev, Renat; Zuo, Wu-Lin; Chao, Ionwa; Fukui, Tomoya; Witover, Bradley; Brekman, Angelika; Crystal, Ronald G

    2013-07-16

    The airway epithelium of smokers acquires pathological phenotypes, including basal cell (BC) and/or goblet cell hyperplasia, squamous metaplasia, structural and functional abnormalities of ciliated cells, decreased number of secretoglobin (SCGB1A1)-expressing secretory cells, and a disordered junctional barrier. In this study, we hypothesized that smoking alters airway epithelial structure through modification of BC function via an EGF receptor (EGFR)-mediated mechanism. Analysis of the airway epithelium revealed that EGFR is enriched in airway BCs, whereas its ligand EGF is induced by smoking in ciliated cells. Exposure of BCs to EGF shifted the BC differentiation program toward the squamous and epithelial-mesenchymal transition-like phenotypes with down-regulation of genes related to ciliogenesis, secretory differentiation, and markedly reduced junctional barrier integrity, mimicking the abnormalities present in the airways of smokers in vivo. These data suggest that activation of EGFR in airway BCs by smoking-induced EGF represents a unique mechanism whereby smoking can alter airway epithelial differentiation and barrier function. PMID:23818594

  4. Phenotypic Profiling of Scedosporium aurantiacum, an Opportunistic Pathogen Colonizing Human Lungs

    PubMed Central

    Kaur, Jashanpreet; Duan, Shu Yao; Vaas, Lea A. I.; Penesyan, Anahit; Meyer, Wieland; Paulsen, Ian T.; Nevalainen, Helena

    2015-01-01

    Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains. PMID:25811884

  5. Visual phenotype in Williams-Beuren syndrome challenges magnocellular theories explaining human neurodevelopmental visual cortical disorders

    PubMed Central

    Castelo-Branco, Miguel; Mendes, Mafalda; Sebastião, Ana Raquel; Reis, Aldina; Soares, Mário; Saraiva, Jorge; Bernardes, Rui; Flores, Raquel; Pérez-Jurado, Luis; Silva, Eduardo

    2007-01-01

    Williams-Beuren syndrome (WBS), a neurodevelopmental genetic disorder whose manifestations include visuospatial impairment, provides a unique model to link genetically determined loss of neural cell populations at different levels of the nervous system with neural circuits and visual behavior. Given that several of the genes deleted in WBS are also involved in eye development and the differentiation of retinal layers, we examined the retinal phenotype in WBS patients and its functional relation to global motion perception. We discovered a low-level visual phenotype characterized by decreased retinal thickness, abnormal optic disk concavity, and impaired visual responses in WBS patients compared with age-matched controls by using electrophysiology, confocal and coherence in vivo imaging with cellular resolution, and psychophysics. These mechanisms of impairment are related to the magnocellular pathway, which is involved in the detection of temporal changes in the visual scene. Low-level magnocellular performance did not predict high-level deficits in the integration of motion and 3D information at higher levels, thereby demonstrating independent mechanisms of dysfunction in WBS that will require remediation strategies different from those used in other visuospatial disorders. These findings challenge neurodevelopmental theories that explain cortical deficits based on low-level magnocellular impairment, such as regarding dyslexia. PMID:18037993

  6. SIK inhibition in human myeloid cells modulates TLR and IL-1R signaling and induces an anti-inflammatory phenotype.

    PubMed

    Lombardi, Maria Stella; Gilliéron, Corine; Dietrich, Damien; Gabay, Cem

    2016-05-01

    Macrophage polarization into a phenotype producing high levels of anti-inflammatory IL-10 and low levels of proinflammatory IL-12 and TNF-α cytokines plays a pivotal role in the resolution of inflammation. Salt-inducible kinases synergize with TLR signaling to restrict the formation of these macrophages. The expression and function of salt-inducible kinase in primary human myeloid cells are poorly characterized. Here, we demonstrated that the differentiation from peripheral blood monocytes to macrophages or dendritic cells induced a marked up-regulation of salt-inducible kinase protein expression. With the use of 2 structurally unrelated, selective salt-inducible kinase inhibitors, HG-9-91-01 and ARN-3236, we showed that salt-inducible kinase inhibition significantly decreased proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-12p40) and increased IL-10 secretion by human myeloid cells stimulated with TLR2 and-4 agonists. Differently than in mouse cells, salt-inducible kinase inhibition did not enhance IL-1Ra production in human macrophages. Salt-inducible kinase inhibition blocked several markers of proinflammatory (LPS + IFN-γ)-polarized macrophages [M(LPS + IFN-γ)] and induced a phenotype characterized by low TNF-α/IL-6/IL-12p70 and high IL-10. The downstream effects observed with salt-inducible kinase inhibitors on cytokine modulation correlated with direct salt-inducible kinase target (CREB-regulated transcription coactivator 3 and histone deacetylase 4) dephosphorylation in these cells. More importantly, we showed for the first time that salt-inducible kinase inhibition decreases proinflammatory cytokines in human myeloid cells upon IL-1R stimulation. Altogether, our results expand the potential therapeutic use of salt-inducible kinase inhibitors in immune-mediated inflammatory diseases. PMID:26590148

  7. Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1.

    PubMed Central

    Clark, R M; Marker, P C; Roessler, E; Dutra, A; Schimenti, J C; Muenke, M; Kingsley, D M

    2001-01-01

    The major locus for dominant preaxial polydactyly in humans has been mapped to 7q36. In mice the dominant Hemimelic extra toes (Hx) and Hammertoe (Hm) mutations map to a homologous chromosomal region and cause similar limb defects. The Lmbr1 gene is entirely within the small critical intervals recently defined for both the mouse and human mutations and is misexpressed at the exact time that the mouse Hx phenotype becomes apparent during limb development. This result suggests that Lmbr1 may underlie preaxial polydactyly in both mice and humans. We have used deletion chromosomes to demonstrate that the dominant mouse and human limb defects arise from gain-of-function mutations and not from haploinsufficiency. Furthermore, we created a loss-of-function mutation in the mouse Lmbr1 gene that causes digit number reduction (oligodactyly) on its own and in trans to a deletion chromosome. The loss of digits that we observed in mice with reduced Lmbr1 activity is in contrast to the gain of digits observed in Hx mice and human polydactyly patients. Our results suggest that the Lmbr1 gene is required for limb formation and that reciprocal changes in levels of Lmbr1 activity can lead to either increases or decreases in the number of digits in the vertebrate limb. PMID:11606546

  8. MUTZ-3 derived Langerhans cells in human skin equivalents show differential migration and phenotypic plasticity after allergen or irritant exposure

    SciTech Connect

    Kosten, Ilona J.; Spiekstra, Sander W.; Gruijl, Tanja D. de; Gibbs, Susan

    2015-08-15

    After allergen or irritant exposure, Langerhans cells (LC) undergo phenotypic changes and exit the epidermis. In this study we describe the unique ability of MUTZ-3 derived Langerhans cells (MUTZ-LC) to display similar phenotypic plasticity as their primary counterparts when incorporated into a physiologically relevant full-thickness skin equivalent model (SE-LC). We describe differences and similarities in the mechanisms regulating LC migration and plasticity upon allergen or irritant exposure. The skin equivalent consisted of a reconstructed epidermis containing primary differentiated keratinocytes and CD1a{sup +} MUTZ-LC on a primary fibroblast-populated dermis. Skin equivalents were exposed to a panel of allergens and irritants. Topical exposure to sub-toxic concentrations of allergens (nickel sulfate, resorcinol, cinnamaldehyde) and irritants (Triton X-100, SDS, Tween 80) resulted in LC migration out of the epidermis and into the dermis. Neutralizing antibody to CXCL12 blocked allergen-induced migration, whereas anti-CCL5 blocked irritant-induced migration. In contrast to allergen exposure, irritant exposure resulted in cells within the dermis becoming CD1a{sup −}/CD14{sup +}/CD68{sup +} which is characteristic of a phenotypic switch of MUTZ-LC to a macrophage-like cell in the dermis. This phenotypic switch was blocked with anti-IL-10. Mechanisms previously identified as being involved in LC activation and migration in native human skin could thus be reproduced in the in vitro constructed skin equivalent model containing functional LC. This model therefore provides a unique and relevant research tool to study human LC biology in situ under controlled in vitro conditions, and will provide a powerful tool for hazard identification, testing novel therapeutics and identifying new drug targets. - Highlights: • MUTZ-3 derived Langerhans cells integrated into skin equivalents are fully functional. • Anti-CXCL12 blocks allergen-induced MUTZ-LC migration.

  9. A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging.

    PubMed

    Patterson, Victoria L; Thompson, Brian S; Cherry, Catherine; Wang, Shao-Bin; Chen, Bo; Hoh, Josephine

    2016-01-01

    Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results

  10. Defect of Fc receptors and phenotypical changes in sinusoidal endothelial cells in human liver cirrhosis.

    PubMed Central

    Muro, H.; Shirasawa, H.; Kosugi, I.; Nakamura, S.

    1993-01-01

    To analyze the pathological changes occurring in Fc receptors (FcRs) in sinusoidal endothelial cells (SECs) in chronic liver diseases, we first characterized immunohistochemically the SEC FcRs by using monoclonal antibodies (MAbs) to FcRs and then investigated the distribution of the SEC FcRs by using peroxidase-antiperoxidase IgG complexes as a ligand on frozen sections. MAb 2E1 to FcRII reacted with SECs in a similar manner to peroxidase-antiperoxidase IgG and blocked the peroxidase-antiperoxidase IgG binding to SECs, whereas MAbs 3G8 and Leu-11b to FcRIII did not. FcRs in normal liver were found along the sinusoidal walls, except for those in the outer periportal zones, but FcRs in chronic active hepatitis and cirrhosis were intermittently or focally absent. The lengths of the FcR-positive portion of sinusoids in unit areas were respectively about 54% and 76% of the normal values in active and inactive cirrhosis. Where FcRs were absent, the MAbs CD36, CD31, and EN4 revealed the presence of sinusoids and, in active cirrhosis, frequently the thickening of liver cell plates. The FcR-negative SECs in the outer periportal zones of normal livers were different from the SECs of other sites in the presence of PAL-E antigen and a rich amount of EN4 antigen, though these sinusoids possessed Kupffer cells and no perisinusoidal deposition of laminin. The FcR-negative SECs in liver diseases occasionally presented the character of ordinary blood vessels, viz., PAL-E antigen, CD34 antigen, and a deficiency of Kupffer cells, regardless of perisinusoidal laminin deposition. However, they preserved the character of normally FcR-possessing SECs, viz., CD36 antigen, and a small amount of EN4 and CD31 antigens. These findings indicate that the outer-periportal SECs in normal livers are phenotypically different from other SECs and that the SECs in diseased livers frequently undergo phenotypical changes, including loss of FcRs, regardless of perisinusoidal laminin deposition, i

  11. Phenotypic Description and Antimicrobial Susceptibilities of Aerococcus sanguinicola Isolates from Human Clinical Samples

    PubMed Central

    Facklam, Richard; Lovgren, Marguerite; Shewmaker, Patricia Lynn; Tyrrell, Gregory

    2003-01-01

    This report describes the clinical sources and phenotypic characterization of 16 isolates of Aerococcus sanguinicola. Sixteen conventional tests were used to describe and differentiate the 16 isolates of A. sanguinicola from 30 strains of Aerococcus viridans, 27 strains of Aerococcus urinae, and a single strain each of Aerococcus christensenii and Aerococcus urinaehominis. The phenotypic characterizations of the type strains for each species and 14 A. sanguinicola isolates were also compared in the two reference laboratories. A. sanguinicola are catalase-negative, vancomycin-susceptible, gram-positive cocci arranged in clusters and tetrads, as are all Aerococcus species except A. christensenii (which is arranged in short chains). All 16 isolates of A. sanguinicola were leucine aminopeptidase and pyrrolidonylarylamidase positive, which is unique to this species among the aerococci. All A. sanguinicola isolates grew in broth containing 6.5% NaCl, hydrolyzed hippurate, and were variable in the bile-esculin test. None of the isolates deaminated arginine or were Voges-Proskauer positive. The type strain of A. sanguinicola was isolated from a blood culture of a patient living in Denmark. Seven additional isolates were from patients living in Canada, all with urinary tract infections (six were female). Eight isolates were from patients living in five different states in the United States; five were from patients with urinary tract infections, and three were from blood cultures of one patient each with pneumonia, suspected endocarditis, and unknown clinical conditions. The antimicrobial susceptibility patterns were unremarkable; all isolates tested were susceptible to penicillin, amoxicillin, cefotaxime, cefuroxime, erythromycin, chloramphenicol, vancomycin, quinupristin-dalfopristin (Synercid), rifampin, linezolid, and tetracycline. Six of the 15 cultures were resistant to ciprofloxacin and levofloxacin, but all 15 strains were susceptible to sparfloxacin. High

  12. Phenotypic description and antimicrobial susceptibilities of Aerococcus sanguinicola isolates from human clinical samples.

    PubMed

    Facklam, Richard; Lovgren, Marguerite; Shewmaker, Patricia Lynn; Tyrrell, Gregory

    2003-06-01

    This report describes the clinical sources and phenotypic characterization of 16 isolates of Aerococcus sanguinicola. Sixteen conventional tests were used to describe and differentiate the 16 isolates of A. sanguinicola from 30 strains of Aerococcus viridans, 27 strains of Aerococcus urinae, and a single strain each of Aerococcus christensenii and Aerococcus urinaehominis. The phenotypic characterizations of the type strains for each species and 14 A. sanguinicola isolates were also compared in the two reference laboratories. A. sanguinicola are catalase-negative, vancomycin-susceptible, gram-positive cocci arranged in clusters and tetrads, as are all Aerococcus species except A. christensenii (which is arranged in short chains). All 16 isolates of A. sanguinicola were leucine aminopeptidase and pyrrolidonylarylamidase positive, which is unique to this species among the aerococci. All A. sanguinicola isolates grew in broth containing 6.5% NaCl, hydrolyzed hippurate, and were variable in the bile-esculin test. None of the isolates deaminated arginine or were Voges-Proskauer positive. The type strain of A. sanguinicola was isolated from a blood culture of a patient living in Denmark. Seven additional isolates were from patients living in Canada, all with urinary tract infections (six were female). Eight isolates were from patients living in five different states in the United States; five were from patients with urinary tract infections, and three were from blood cultures of one patient each with pneumonia, suspected endocarditis, and unknown clinical conditions. The antimicrobial susceptibility patterns were unremarkable; all isolates tested were susceptible to penicillin, amoxicillin, cefotaxime, cefuroxime, erythromycin, chloramphenicol, vancomycin, quinupristin-dalfopristin (Synercid), rifampin, linezolid, and tetracycline. Six of the 15 cultures were resistant to ciprofloxacin and levofloxacin, but all 15 strains were susceptible to sparfloxacin. High

  13. Large animal models of rare genetic disorders: sheep as phenotypically relevant models of human genetic disease.

    PubMed

    Pinnapureddy, Ashish R; Stayner, Cherie; McEwan, John; Baddeley, Olivia; Forman, John; Eccles, Michael R

    2015-01-01

    Animals that accurately model human disease are invaluable in medical research, allowing a critical understanding of disease mechanisms, and the opportunity to evaluate the effect of therapeutic compounds in pre-clinical studies. Many types of animal models are used world-wide, with the most common being small laboratory animals, such as mice. However, rodents often do not faithfully replicate human disease, despite their predominant use in research. This discordancy is due in part to physiological differences, such as body size and longevity. In contrast, large animal models, including sheep, provide an alternative to mice for biomedical research due to their greater physiological parallels with humans. Completion of the full genome sequences of many species, and the advent of Next Generation Sequencing (NGS) technologies, means it is now feasible to screen large populations of domesticated animals for genetic variants that resemble human genetic diseases, and generate models that more accurately model rare human pathologies. In this review, we discuss the notion of using sheep as large animal models, and their advantages in modelling human genetic disease. We exemplify several existing naturally occurring ovine variants in genes that are orthologous to human disease genes, such as the Cln6 sheep model for Batten disease. These, and other sheep models, have contributed significantly to our understanding of the relevant human disease process, in addition to providing opportunities to trial new therapies in animals with similar body and organ size to humans. Therefore sheep are a significant species with respect to the modelling of rare genetic human disease, which we summarize in this review. PMID:26329332

  14. Improving external cephalic version for foetal breech presentation

    PubMed Central

    Zandstra, H.; Mertens, H.J.M.M.

    2013-01-01

    Objectives: If success rate of external cephalic version (ECV) increases, the rate of primary caesarean sections declines. The aims of this retrospective cohort study were to evaluate the ECV and to identify factors associated with the success rate of ECV for breech presentation at term. The second aim of this study was to analyse the outcome of labour of all patients with a foetus in breech near term. Methods: All women with a foetus in breech near or at term were included. Logistic regression analyses were performed to identify the association between patient characteristics and success rate of ECV. Results: The overall rate of successful ECV’s was 19%. Foetal and maternal complications after ECV were negligible. BMI, type of breech and amount of amniotic fluid were significantly correlated with a successful ECV. The rate of primary caesarean sections for the group of patients who underwent an ECV was lower than the rate in the group who did not (52.9% vs. 79.6%). The rate of spontaneous deliveries was increased after ECV (36% versus 12%). After successful ECV the rate of spontaneous deliveries was 75%; after unsuccessful ECV 26.8%. Conclusion: The overall rate of successful ECV was low (19%). BMI, type of breech and amount of amniotic fluid were significantly correlated with a successful ECV. The rate of primary caesarean sections was significantly lower in patients with ECV (52.9% versus 79.6%). The rate of spontaneous deliveries was significantly higher (36% versus 12%). PMID:24753933

  15. Maternal Geophagy of Calabash Chalk on Foetal Cerebral Cortex Histomorphology

    PubMed Central

    EKANEM, Theresa Bassey; EKONG, Moses Bassey; ELUWA, Mokutima Amarachi; IGIRI, Anozeng Oyono; OSIM, Eme Efiom

    2015-01-01

    Background: Calabash chalk, a kaolin-base substance is a common geophagic material mostly consumed by pregnant women. This study investigated its effect on the histomorphology of the foetal cerebral cortex. Methods: Twelve gestating Wistar rats were divided equally into groups 1 and 2. On pregnancy day seven (PD7), group 2 animals were administered 200 mg/kg body weight of calabash chalk suspension, while group 1 animals served as the control and received 1 ml of distilled water, by oral gavages and for 14 days (PD7-PD20). On PD21, the dams were sacrificed, and the foetuses removed, examined for gross malformations, weighed and culled to two foetuses per mother. Their whole brains were excised, weighed and preserved using 10% buffered formalin, and routinely processed by haematoxylin and eosin, and Luxol fast blue methods. Results: The foetuses showed no morphological change, but their mean body weights was higher (p=0.0001). Histomorphological sections of the cerebral cortex showed hypertrophy and hyperplasia of cells in all the cortical layers, with less demonstrated Nissl and higher (p=0.001) cellular population compared with the control group. Conclusion: Calabash chalk cause body weight increase and histomorphological changes in the cerebral cortex of foetuses. PMID:26715904

  16. ZIKA VIRUS INFECTION; VERTICAL TRANSMISSION AND FOETAL CONGENITAL ANOMALIES.

    PubMed

    Abbasi, Aziz-un-Nisa

    2016-01-01

    Zika virus (ZIKV) is an arbovirus belonging to flaviviridae family that includes Dengue, West Nile, and Yellow Fever among others. Zika virus was first discovered in 1947 in Zika forest of Uganda. It is a vector borne disease, which has been sporadically reported mostly from Africa, Pacific islands and Southeast Asia since its discovery. ZIKV infection presents as a mild illness with symptoms lasting for several days to a week after the bite of an infected mosquito. Majority of the patients have low grade fever, rash, headaches, joints pain, myalgia, and flu like symptoms. Pregnant women are more vulnerable to ZIKV infection and serious congenital anomalies can occur in foetus through trans-placental transmission. The gestation at which infection is acquired is important. Zika virus infection acquired in early pregnancy poses greater risk. There is no evidence so far about transmission through breast milk. Foetal microcephaly, Gillian Barre syndrome and other neurological and autoimmune syndromes have been reported in areas where Zika outbreaks have occurred. As infection is usually very mild no specific treatment is required. Pregnant women may be advised to take rest, get plenty of fluids. For fever and pain they can take antipyretics like paracetamol. So far no specific drugs or vaccines are available against Zika Virus Infection so prevention is the mainstay against this diseases. As ZIKV infection is a vector borne disease, prevention can be a multi-pronged strategy. These entail vector control interventions, personal protection, environmental sanitation and health education among others. PMID:27323550

  17. Retinol and Retinyl Palmitate in Foetal Lung Mice: Sexual Dimorphism

    PubMed Central

    Carvalho, Olga; Gonçalves, Carlos

    2013-01-01

    In this work, we evaluate the lung retinoids content to study the possible difference between male and female mice during prenatal development and to comprehend if the vitamin A metabolism is similar in both genders. The study occurred between developmental days E15 and E19, and the retinol and retinyl palmitate lung contents were determined by HPLC analysis. We established two main groups: the control, consisting of foetuses obtained from pregnant females without any manipulation, and vitamin A, composed of foetuses from pregnant females submitted to vitamin A administration on developmental day E14. Each of these groups was subdivided by gender, establishing the four final groups. In the lung of control group, retinol was undetected in both genders and retinyl palmitate levels exhibited a sexual dimorphism. In the vitamin A group, we detected retinol and retinyl palmitate in both genders, and we observed a more evident sexual dimorphism for both retinoids. Our study also indicates that, from developmental day E15 to E19, there is an increase in the retinoids content in foetal lung and a gender difference in the retinoids metabolism. In conclusion, there is a sexual dimorphism in the lung retinoids content and in its metabolism during mice development. PMID:23365730

  18. Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

    PubMed

    Della Chiesa, Mariella; Falco, Michela; Podestà, Marina; Locatelli, Franco; Moretta, Lorenzo; Frassoni, Francesco; Moretta, Alessandro

    2012-01-12

    Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR(+)NKG2A(-) signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C(+) NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56(-)CD16(+)p75/AIRM1(-) NK-cell subset (mostly KIR(+)NKG2A(-)) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation. PMID:22096237

  19. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons.

    PubMed

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P

    2015-11-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to

  20. Cosmetics as a feature of the extended human phenotype: modulation of the perception of biologically important facial signals.

    PubMed

    Etcoff, Nancy L; Stock, Shannon; Haley, Lauren E; Vickery, Sarah A; House, David M

    2011-01-01

    Research on the perception of faces has focused on the size, shape, and configuration of inherited features or the biological phenotype, and largely ignored the effects of adornment, or the extended phenotype. Research on the evolution of signaling has shown that animals frequently alter visual features, including color cues, to attract, intimidate or protect themselves from conspecifics. Humans engage in conscious manipulation of visual signals using cultural tools in real time rather than genetic changes over evolutionary time. Here, we investigate one tool, the use of color cosmetics. In two studies, we asked viewers to rate the same female faces with or without color cosmetics, and we varied the style of makeup from minimal (natural), to moderate (professional), to dramatic (glamorous). Each look provided increasing luminance contrast between the facial features and surrounding skin. Faces were shown for 250 ms or for unlimited inspection time, and subjects rated them for attractiveness, competence, likeability and trustworthiness. At 250 ms, cosmetics had significant positive effects on all outcomes. Length of inspection time did not change the effect for competence or attractiveness. However, with longer inspection time, the effect of cosmetics on likability and trust varied by specific makeup looks, indicating that cosmetics could impact automatic and deliberative judgments differently. The results suggest that cosmetics can create supernormal facial stimuli, and that one way they may do so is by exaggerating cues to sexual dimorphism. Our results provide evidence that judgments of facial trustworthiness and attractiveness are at least partially separable, that beauty has a significant positive effect on judgment of competence, a universal dimension of social cognition, but has a more nuanced effect on the other universal dimension of social warmth, and that the extended phenotype significantly influences perception of biologically important signals at first

  1. Cosmetics as a Feature of the Extended Human Phenotype: Modulation of the Perception of Biologically Important Facial Signals

    PubMed Central

    Etcoff, Nancy L.; Stock, Shannon; Haley, Lauren E.; Vickery, Sarah A.; House, David M.

    2011-01-01

    Research on the perception of faces has focused on the size, shape, and configuration of inherited features or the biological phenotype, and largely ignored the effects of adornment, or the extended phenotype. Research on the evolution of signaling has shown that animals frequently alter visual features, including color cues, to attract, intimidate or protect themselves from conspecifics. Humans engage in conscious manipulation of visual signals using cultural tools in real time rather than genetic changes over evolutionary time. Here, we investigate one tool, the use of color cosmetics. In two studies, we asked viewers to rate the same female faces with or without color cosmetics, and we varied the style of makeup from minimal (natural), to moderate (professional), to dramatic (glamorous). Each look provided increasing luminance contrast between the facial features and surrounding skin. Faces were shown for 250 ms or for unlimited inspection time, and subjects rated them for attractiveness, competence, likeability and trustworthiness. At 250 ms, cosmetics had significant positive effects on all outcomes. Length of inspection time did not change the effect for competence or attractiveness. However, with longer inspection time, the effect of cosmetics on likability and trust varied by specific makeup looks, indicating that cosmetics could impact automatic and deliberative judgments differently. The results suggest that cosmetics can create supernormal facial stimuli, and that one way they may do so is by exaggerating cues to sexual dimorphism. Our results provide evidence that judgments of facial trustworthiness and attractiveness are at least partially separable, that beauty has a significant positive effect on judgment of competence, a universal dimension of social cognition, but has a more nuanced effect on the other universal dimension of social warmth, and that the extended phenotype significantly influences perception of biologically important signals at first

  2. Hypoxia-derived oxidative stress mediates epigenetic repression of PKCɛ gene in foetal rat hearts

    PubMed Central

    Patterson, Andrew J.; Xiao, Daliao; Xiong, Fuxia; Dixon, Brandon; Zhang, Lubo

    2012-01-01

    Aims Hypoxia causes protein kinase C epsilon (PKCɛ) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCɛ gene repression. Methods and results Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCɛ protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCɛ promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCɛ mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCɛ protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCɛ promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring. Conclusions The results demonstrate that hypoxia induces epigenetic repression of the PKCɛ gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring. PMID:22139554

  3. RasGRP3, a Ras activator, contributes to signaling and the tumorigenic phenotype in human melanoma

    PubMed Central

    Li, Luowei; Kedei, Noemi; Tóth, Zsuzsanna E.; Czap, Alexandra; Velasquez, Julia F.; Mihova, Daniela; Michalowski, Aleksandra M.; Yuspa, Stuart H.; Blumberg, Peter M.

    2014-01-01

    RasGRP3, an activator for H-Ras, R-Ras and Rap1/2, has emerged as an important mediator of signaling downstream from receptor coupled phosphoinositide turnover in B and T cells. Here, we report that RasGRP3 showed a high level of expression in multiple human melanoma cell lines as well as in a subset of human melanoma tissue samples. Suppression of endogenous RasGRP3 expression in these melanoma cell lines reduced Ras-GTP formation as well as c-Met expression and Akt phosphorylation downstream from HGF or EGF stimulation. RasGRP3 suppression also inhibited cell proliferation and reduced both colony formation in soft agar and xenograft tumor growth in immunodeficient mice, demonstrating the importance of RasGRP3 for the transformed phenotype of the melanoma cells. Reciprocally, overexpression of RasGRP3 in human primary melanocytes altered cellular morphology, markedly enhanced cell proliferation, and rendered the cells tumorigenic in a mouse xenograft model. Suppression of RasGRP3 expression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. The identification of the role of RasGRP3 in melanoma highlights its importance, as a Ras activator, in the phosphoinositide signaling pathway in human melanoma and provides a new potential therapeutic target. PMID:21602881

  4. Multiparametric Phenotypic Screening System for Profiling Bioactive Compounds Using Human Fetal Hippocampal Neural Stem/Progenitor Cells.

    PubMed

    Tabata, Yoshikuni; Murai, Norio; Sasaki, Takeo; Taniguchi, Sachie; Suzuki, Shuichi; Yamazaki, Kazuto; Ito, Masashi

    2015-10-01

    Stem cell research has been progressing rapidly, contributing to regenerative biology and regenerative medicine. In this field, small-molecule compounds affecting stem cell proliferation/differentiation have been explored to understand stem cell biology and support regenerative medicine. In this study, we established a multiparametric screening system to detect bioactive compounds affecting the cell fate of human neural stem/progenitor cells (NSCs/NPCs), using human fetal hippocampal NSCs/NPCs, HIP-009 cells. We examined effects of 410 compounds, which were collected based on mechanisms of action (MOAs) and chemotypes, on HIP-009's cell fate (self-renewal, neuronal and astrocytic differentiation) and morphology by automated multiparametric assays and profiled induced cellular phenotypes. We found that this screening classified compounds with the same MOAs into subgroups according to additional pharmacological effects (e.g., mammalian target of rapamycin complex 1 [mTORC1] inhibitors and mTORC1/mTORC2 dual inhibitors among mTOR inhibitors). Moreover, it identified compounds that have off-target effects under matrix analyses of MOAs and structure similarities (e.g., neurotropic effects of amitriptyline among tri- and tetracyclic compounds). Therefore, this automated, medium-throughput and multiparametric screening system is useful for finding compounds that affect the cell fate of human NSCs/NPCs for supporting regenerative medicine and to fingerprint compounds based on human stem cells' multipotency, leading to understanding of stem cell biology. PMID:26245650

  5. Proteome dynamics during contractile and metabolic differentiation of bovine foetal muscle.

    PubMed

    Chaze, T; Meunier, B; Chambon, C; Jurie, C; Picard, B

    2009-07-01

    Contractile and metabolic properties of bovine muscles play an important role in meat sensorial quality, particularly tenderness. Earlier studies based on Myosin heavy chain isoforms analyses and measurements of glycolytic and oxidative enzyme activities have demonstrated that the third trimester of foetal life in bovine is characterized by contractile and metabolic differentiation. In order to complete this data and to obtain a precise view of this phase and its regulation, we performed a proteomic analysis of Semitendinosus muscle from Charolais foetuses analysed at three stages of the third trimester of gestation (180, 210 and 260 days). The results complete the knowledge of important changes in the profiles of proteins from metabolic and contractile pathways. They provide new insights about proteins such as Aldehyde dehydrogenase family, Enolase, Dihydrolipoyl dehydrogenase, Troponin T or Myosin light chains isoforms. These data have agronomical applications not only for the management of beef sensorial quality but also in medical context, as bovine myogenesis appears very similar to human one. PMID:22444818

  6. Drosophila expressing human SOD1 successfully recapitulates mitochondrial phenotypic features of familial amyotrophic lateral sclerosis.

    PubMed

    Gallart-Palau, Xavier; Ng, Chee-Hoe; Ribera, Joan; Sze, Siu Kwan; Lim, Kah-Leong

    2016-06-15

    Mitochondrial pathology is a seminal pathogenic hallmark of familial amyotrophic lateral sclerosis (FALS) which is extensively manifested by human patients and mutant SOD1(G93A) mammalian models. Rodents expressing human FALS-associated mutations successfully mimic several human disease features; although they are not as amenable to genetic and therapeutic compound screenings as non-mammalian models. In this study, we report a newly generated and characterized Drosophila model that expresses human SOD1(G93A) in muscle fibers. Presence of SOD1(G93A) in thoracic muscles causes mitochondrial pathology and impairs normal motor behavior in these flies. Use of this new FALS-24B-SOD1(G93A) fly model holds promise for better understanding of the mitochondrial affectation process in FALS and for the discovery of novel therapeutic compounds able to reverse mitochondrial dysfunction in this fatal disease. PMID:27163198

  7. Release of Tensile Strain on Engineered Human Tendon Tissue Disturbs Cell Adhesions, Changes Matrix Architecture, and Induces an Inflammatory Phenotype

    PubMed Central

    Bayer, Monika L.; Schjerling, Peter; Herchenhan, Andreas; Zeltz, Cedric; Heinemeier, Katja M.; Christensen, Lise; Krogsgaard, Michael; Gullberg, Donald; Kjaer, Michael

    2014-01-01

    Mechanical loading of tendon cells results in an upregulation of mechanotransduction signaling pathways, cell-matrix adhesion and collagen synthesis, but whether unloading removes these responses is unclear. We investigated the response to tension release, with regard to matrix proteins, pro-inflammatory mediators and tendon phenotypic specific molecules, in an in vitro model where tendon-like tissue was engineered from human tendon cells. Tissue sampling was performed 1, 2, 4 and 6 days after surgical de-tensioning of the tendon construct. When tensile stimulus was removed, integrin type collagen receptors showed a contrasting response with a clear drop in integrin subunit α11 mRNA and protein expression, and an increase in α2 integrin mRNA and protein levels. Further, specific markers for tendon cell differentiation declined and normal tendon architecture was disturbed, whereas pro-inflammatory molecules were upregulated. Stimulation with the cytokine TGF-β1 had distinct effects on some tendon-related genes in both tensioned and de-tensioned tissue. These findings indicate an important role of mechanical loading for cellular and matrix responses in tendon, including that loss of tension leads to a decrease in phenotypical markers for tendon, while expression of pro-inflammatory mediators is induced. PMID:24465881

  8. Human airway smooth muscle maintain in situ cell orientation and phenotype when cultured on aligned electrospun scaffolds

    PubMed Central

    Morris, G. E.; Bridge, J. C.; Eltboli, O. M. I.; Lewis, M. P.; Knox, A. J.; Aylott, J. W.; Brightling, C. E.; Ghaemmaghami, A. M.

    2014-01-01

    Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared with 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibers produced have dimensions within the nanometer range, similar to cells' natural environment. We have developed an electrospun scaffold using the nondegradable, nontoxic, polymer polyethylene terephthalate (PET) composed of uniaxially orientated nanofibers and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibers orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development toward a more in vivo-like phenotype while providing topological cues to ensure full cell alignment. PMID:24793171

  9. Mutations in CSPP1, Encoding a Core Centrosomal Protein, Cause a Range of Ciliopathy Phenotypes in Humans

    PubMed Central

    Shaheen, Ranad; Shamseldin, Hanan E.; Loucks, Catrina M.; Seidahmed, Mohammed Zain; Ansari, Shinu; Ibrahim Khalil, Mohamed; Al-Yacoub, Nadya; Davis, Erica E.; Mola, Natalie A.; Szymanska, Katarzyna; Herridge, Warren; Chudley, Albert E.; Chodirker, Bernard N.; Schwartzentruber, Jeremy; Majewski, Jacek; Katsanis, Nicholas; Poizat, Coralie; Johnson, Colin A.; Parboosingh, Jillian; Boycott, Kym M.; Innes, A. Micheil; Alkuraya, Fowzan S.

    2014-01-01

    Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies. PMID:24360803

  10. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections.

    PubMed

    Meier, Ute-Christiane; Owen, Rachel E; Taylor, Elizabeth; Worth, Andrew; Naoumov, Nikolai; Willberg, Christian; Tang, Kwok; Newton, Phillipa; Pellegrino, Pierre; Williams, Ian; Klenerman, Paul; Borrow, Persephone

    2005-10-01

    Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections. PMID:16160163

  11. [The reproductive characteristics of human adenovirus type 2 in cultures of lymphoblastoid cells with B and T phenotypes].

    PubMed

    Povnitsa, O Iu; Diachenko, N S; Chernomaz, A A; Nosach, L N; Rybalko, S L; Gritsak, T F; Beregovenko, V N; Diadiun, S T

    1997-01-01

    A comparative characteristic of the reproduction process of type 2 human adenovirus in several lines of lymphoblastoid cells of B- and T-phenotype is presented. Formation of hexone and infectious virus in the cells of Jurkat, MT4, Raji lines was rather intensive and approached to that in the culture of the permissive epithelium cells Hep-2. These indices were much lower in the cultures of cells B 95-8 and MT4/BIII LBK which were chronically infected by VEB and HIV, accordingly and produced them that can evidence for the interference of Ad and VEB or Ad and HIV under superinfection of cells. Cells of SEM line possessing T-phenotype, were apparently semi-permissive for Ad h2, though the low almost unchanged content of hexone and infectious virus remains in them for a rather long time: about 15 days. Thus, obtained data within analyzed series of experiments expand the present ideas about lymphotropicity of Ad as their important property realized at the level of cell and infected macroorganism. PMID:9511371

  12. Genotypic and phenotypic characterization of Helicobacter cinaedi and Helicobacter fennelliae strains isolated from humans and animals.

    PubMed Central

    Kiehlbauch, J A; Brenner, D J; Cameron, D N; Steigerwalt, A G; Makowski, J M; Baker, C N; Patton, C M; Wachsmuth, I K

    1995-01-01

    By DNA-DNA hybridization, we classified 26 human strains, 4 dog and cat strains, and 4 hamster strains putatively identified as Helicobacter cinaedi as well as 2 human strains and 2 animal strains of Helicobacter fennelliae. All but one human strain belonged to the same hybridization group as the type strain of H. cinaedi. The animal strains also appeared to belong to this hybridization group. Both human strains of H. fennelliae were shown to be H. fennelliae by DNA-DNA hybridization, but both animal strains were less than 15% related to the type strain. All strains were also characterized by plasmid profiles and ribotyping. Plasmids were found in 23% of the human strains, 100% of the hamster strains, and 33% of the dog and cat strains. Human strains were essentially identical by ribotyping, but were clearly differentiated from the hamster and dog and cat strains. Some strains may be difficult to culture on primary isolation; we found that our strains grew well on anaerobic CDC agar, brucella agar, and tryptic soy agar II. Our H. cinaedi and H. fennelliae strains differed from those previously described because some were resistant to cephalothin: some H. cinaedi strains were also resistant to nalidixic acid. All isolates were also characterized by antimicrobial susceptibility testing. We found that human strains of H. cinaedi were more resistant to clindamycin and erythromycin than were animal isolates; 19% of the human strains were resistant to ciprofloxacin. Therefore, we recommend that antimicrobial susceptibility results be obtained before initiating therapy for H. cinaedi and H. fennelliae infections. PMID:8576350

  13. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells

    SciTech Connect

    Grant, D.J.; Maeda, N. )

    1993-05-01

    An A-to-C base substitution at nucleotide position -61 in the promoter region of the human haptoglobin gene (Hp) has been shown to be strongly associated with the haptoglobin 2-1 modified (Hp2-1mod) phenotype. In order to investigate whether this base substitution is the cause of reduced expression of the Hp[sup 2] allele relative to the Hp[sup 1] allele in individuals with the Hp2-1mod phenotype, the authors used the chloramphenicol acetyl transferase (CAT) expression system to evaluate promoter function. In HepG2 cells, which normally express their endogenous haptoglobin genes, CAT plasmid constructs with the -61C base change in the promoter had about 10-fold-lower transcriptional activity after transfection than did the Hp control construct. The -61C substitution also rendered the construct unresponsive to treatment by interleukin-6 after transfection into Hep3B2 cells, which normally do not express haptoglobin but do so in response to stimulation by acute-phase reactants. In addition, two base substitutions, T to A and A to G, at positions -104 and -55G, respectively, in the promoter region of the Hp[sup 1] allele, are also associated with the Hp2-1mod phenotype. CAT constructs with both substitutions (-104A-55G) and with one substitution (-55G) showed activity similar to that in the Hp control when transfected into both HepG2 and Hep3B2 cells, although interleukin-6 induction was less than with the Hp control construct. These results further support the hypothesis that the Hp2-1mod phenotype results, in part, from the -61C mutation in the promoter region of the Hp[sup 2] gene.

  14. Prognostic value of Dicer expression in human breast cancers and association with the mesenchymal phenotype

    PubMed Central

    Grelier, G; Voirin, N; Ay, A-S; Cox, D G; Chabaud, S; Treilleux, I; Léon-Goddard, S; Rimokh, R; Mikaelian, I; Venoux, C; Puisieux, A; Lasset, C; Moyret-Lalle, C

    2009-01-01

    Background: Dicer, a ribonuclease, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs and is essential for both mammalian development and cell differentiation. Recent evidence indicates that Dicer may also be involved in tumourigenesis. However, no studies have examined the clinical significance of Dicer at both the RNA and the protein levels in breast cancer. Methods: In this study, the biological and prognostic value of Dicer expression was assessed in breast cancer cell lines, breast cancer progression cellular models, and in two well-characterised sets of breast carcinoma samples obtained from patients with long-term follow-up using tissue microarrays and quantitative reverse transcription–PCR. Results: We have found that Dicer protein expression is significantly associated with hormone receptor status and cancer subtype in breast tumours (ER P=0.008; PR P=0.019; cancer subtype P=0.023, luminal A P=0.0174). Dicer mRNA expression appeared to have an independent prognostic impact in metastatic disease (hazard ratio=3.36, P=0.0032). In the breast cancer cell lines, lower Dicer expression was found in cells harbouring a mesenchymal phenotype and in metastatic bone derivatives of a breast cancer cell line. These findings suggest that the downregulation of Dicer expression may be related to the metastatic spread of tumours. Conclusion: Assessment of Dicer expression may facilitate prediction of distant metastases for patients suffering from breast cancer. PMID:19672267

  15. Phenotyping of Live Human PBMC using CyTOF™ Mass Cytometry

    PubMed Central

    Leipold, Michael D.; Maecker, Holden T.

    2016-01-01

    Single-cell analysis has become an method of importance in immunology. Fluorescence flow cytometry has been a major player. However, due to issues such as autofluorescence and emission spillover between different fluorophores, alternative techniques are being developed. In recent years, mass cytometry has emerged, wherein antibodies labeled with metal ions are detected by ICP-MS. In order for a cell to be seen, a metal in the mass window must be present; there is no analogous parameter to forward or side scatter. The current mass window selected is approximately AW 103-196, which includes the lanthanides used for most antibody labeling, as well as iridium and rhodium for DNA intercalators. In this protocol, we use a cocktail of antibodies labeled with MAXPAR metal-chelating polymers to surface-stain live PBMC that have been previously cryopreserved. Many of these markers were taken from a standard fluorescence phenotyping panel (Maecker et al., 2012). No intracellular antibodies are used. We use a CyTOF™ (Cytometry by Time-Of-Flight) mass cytometer to acquire the ICP-MS data. Subsequent analysis of the dual count signal data using FlowJo software allows for cell types to be analyzed based on the dual count signal in each mass channel. The percentage of each cell type is determined and reported as a percent of the parent cell type.

  16. Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data.

    PubMed

    Dewey, Frederick E; Grove, Megan E; Priest, James R; Waggott, Daryl; Batra, Prag; Miller, Clint L; Wheeler, Matthew; Zia, Amin; Pan, Cuiping; Karzcewski, Konrad J; Miyake, Christina; Whirl-Carrillo, Michelle; Klein, Teri E; Datta, Somalee; Altman, Russ B; Snyder, Michael; Quertermous, Thomas; Ashley, Euan A

    2015-10-01

    High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework. PMID:26448358

  17. Sequence to Medical Phenotypes: A Framework for Interpretation of Human Whole Genome DNA Sequence Data

    PubMed Central

    Dewey, Frederick E.; Grove, Megan E.; Priest, James R.; Waggott, Daryl; Batra, Prag; Miller, Clint L.; Wheeler, Matthew; Zia, Amin; Pan, Cuiping; Karzcewski, Konrad J.; Miyake, Christina; Whirl-Carrillo, Michelle; Klein, Teri E.; Datta, Somalee; Altman, Russ B.; Snyder, Michael; Quertermous, Thomas; Ashley, Euan A.

    2015-01-01

    Abstract High throughput sequencing has facilitated a precipitous drop in the cost of genomic sequencing, prompting predictions of a revolution in medicine via genetic personalization of diagnostic and therapeutic strategies. There are significant barriers to realizing this goal that are related to the difficult task of interpreting personal genetic variation. A comprehensive, widely accessible application for interpretation of whole genome sequence data is needed. Here, we present a series of methods for identification of genetic variants and genotypes with clinical associations, phasing genetic data and using Mendelian inheritance for quality control, and providing predictive genetic information about risk for rare disease phenotypes and response to pharmacological therapy in single individuals and father-mother-child trios. We demonstrate application of these methods for disease and drug response prognostication in whole genome sequence data from twelve unrelated adults, and for disease gene discovery in one father-mother-child trio with apparently simplex congenital ventricular arrhythmia. In doing so we identify clinically actionable inherited disease risk and drug response genotypes in pre-symptomatic individuals. We also nominate a new candidate gene in congenital arrhythmia, ATP2B4, and provide experimental evidence of a regulatory role for variants discovered using this framework. PMID:26448358

  18. Mutations in the human UBR1 gene and the associated phenotypic spectrum.

    PubMed

    Sukalo, Maja; Fiedler, Ariane; Guzmán, Celina; Spranger, Stephanie; Addor, Marie-Claude; McHeik, Jiad N; Oltra Benavent, Manuel; Cobben, Jan M; Gillis, Lynette A; Shealy, Amy G; Deshpande, Charu; Bozorgmehr, Bita; Everman, David B; Stattin, Eva-Lena; Liebelt, Jan; Keller, Klaus-Michael; Bertola, Débora Romeo; van Karnebeek, Clara D M; Bergmann, Carsten; Liu, Zhifeng; Düker, Gesche; Rezaei, Nima; Alkuraya, Fowzan S; Oğur, Gönül; Alrajoudi, Abdullah; Venegas-Vega, Carlos A; Verbeek, Nienke E; Richmond, Erick J; Kirbiyik, Ozgür; Ranganath, Prajnya; Singh, Ankur; Godbole, Koumudi; Ali, Fouad A M; Alves, Crésio; Mayerle, Julia; Lerch, Markus M; Witt, Heiko; Zenker, Martin

    2014-05-01

    Johanson-Blizzard syndrome (JBS) is a rare, autosomal recessive disorder characterized by exocrine pancreatic insufficiency, typical facial features, dental anomalies, hypothyroidism, sensorineural hearing loss, scalp defects, urogenital and anorectal anomalies, short stature, and cognitive impairment of variable degree. This syndrome is caused by a defect of the E3 ubiquitin ligase UBR1, which is part of the proteolytic N-end rule pathway. Herein, we review previously reported (n = 29) and a total of 31 novel UBR1 mutations in relation to the associated phenotype in patients from 50 unrelated families. Mutation types include nonsense, frameshift, splice site, missense, and small in-frame deletions consistent with the hypothesis that loss of UBR1 protein function is the molecular basis of JBS. There is an association of missense mutations and small in-frame deletions with milder physical abnormalities and a normal intellectual capacity, thus suggesting that at least some of these may represent hypomorphic UBR1 alleles. The review of clinical data of a large number of molecularly confirmed JBS cases allows us to define minimal clinical criteria for the diagnosis of JBS. For all previously reported and novel UBR1 mutations together with their clinical data, a mutation database has been established at LOVD. PMID:24599544

  19. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    PubMed

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  20. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes

    PubMed Central

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P.; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J.; Gathercole, Laura L.

    2015-01-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  1. Modulation of Invasive Phenotype by Interstitial Pressure-Driven Convection in Aggregates of Human Breast Cancer Cells

    PubMed Central

    Tien, Joe; Truslow, James G.; Nelson, Celeste M.

    2012-01-01

    This paper reports the effect of elevated pressure on the invasive phenotype of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells. We found that the directionality of the interstitial pressure profile altered the frequency of invasion by cells located at the surface of an aggregate. In particular, application of pressure at one end of an aggregate suppressed invasion at the opposite end. Experimental alteration of the configuration of cell aggregates and computational modeling of the resulting flow and solute concentration profiles revealed that elevated pressure inhibited invasion by altering the chemical composition of the interstitial fluid near the surface of the aggregate. Our data reveal a link between hydrostatic pressure, interstitial convection, and invasion. PMID:23028839

  2. An analysis of myeloma plasma cell phenotype using antibodies defined at the IIIrd International Workshop on Human Leucocyte Differentiation Antigens.

    PubMed Central

    Jackson, N; Ling, N R; Ball, J; Bromidge, E; Nathan, P D; Franklin, I M

    1988-01-01

    Fresh bone marrow from 43 cases of myeloma and three cases of plasma cell leukaemia has been phenotyped both by indirect immune-rosetting and, on fixed cytospin preparations, by indirect immunofluorescence. Both clustered and unclustered B cell associated antibodies from the IIIrd International Workshop on Human Leucocyte Differentiation Antigens were used. The results confirm the lack of many pan-B antigens on the surface of myeloma plasma cells, i.e. CD19-23, 37, 39, w40. Strong surface reactivity is seen with CD38 antibodies and with one CD24 antibody (HB8). Weak reactions are sometimes obtained with CD9, 10 and 45R. On cytospin preparations CD37, 39 and w40 are sometimes weakly positive, and anti-rough endoplasmic reticulum antibodies are always strongly positive. Specific and surface-reacting antiplasma cell antibodies are still lacking. PMID:3048803

  3. Genomic and phenotypic evidence for probiotic influences of Lactobacillus gasseri on human health.

    PubMed

    Selle, Kurt; Klaenhammer, Todd R

    2013-11-01

    Certain lactic acid bacteria (LAB) have the capacity to occupy mucosal niches of humans, including the oral cavity, gastrointestinal tract, and vagina. Among commensal, LAB are species of the acidophilus complex, which have proven to be a substantial reservoir for microorganisms with probiotic attributes. Specifically, Lactobacillus gasseri is an autochthonous microorganism which has been evaluated for probiotic activity based on the availability of genome sequence and species-specific adaptation to the human mucosa. Niche-related characteristics of L. gasseri contributing to indigenous colonization include tolerance of low pH environments, resistance to bile salts, and adhesion to the host epithelium. In humans, L. gasseri elicits various health benefits through its antimicrobial activity, bacteriocin production, and immunomodulation of the innate and adaptive systems. The genomic and empirical evidence supporting use of L. gasseri in probiotic applications is substantiated by clinical trial data displaying maintenance of vaginal homeostasis, mitigation of Helicobacter pylori infection, and amelioration of diarrhea. PMID:23488471

  4. Selection for a CEACAM receptor-specific binding phenotype during Neisseria gonorrhoeae infection of the human genital tract.

    PubMed

    Sintsova, Anna; Wong, Henry; MacDonald, Kelly S; Kaul, Rupert; Virji, Mumtaz; Gray-Owen, Scott D

    2015-04-01

    Infections by Neisseria gonorrhoeae are increasingly common, are often caused by antibiotic-resistant strains, and can result in serious and lasting sequelae, prompting the reemergence of gonococcal disease as a leading global health concern. N. gonorrhoeae is a human-restricted pathogen that primarily colonizes urogenital mucosal surfaces. Disease progression varies greatly between the sexes: men usually present with symptomatic infection characterized by a painful purulent urethral discharge, while in women, the infection is often asymptomatic, with the most severe pathology occurring when the bacteria ascend from the lower genital tract into the uterus and fallopian tubes. Classical clinical studies demonstrated that clinically infectious strains uniformly express Opa adhesins; however, their specificities were unknown at the time. While in vitro studies have since identified CEACAM proteins as the primary target of Opa proteins, the gonococcal specificity for this human family of receptors has not been addressed in the context of natural infection. In this study, we characterize a collection of low-passage-number clinical-specimen-derived N. gonorrhoeae isolates for Opa expression and assess their CEACAM-binding profiles. We report marked in vivo selection for expression of phase-variable Opa proteins that bind CEACAM1 and CEACAM5 but selection against expression of Opa variants that bind to the neutrophil-restricted decoy receptor CEACAM3. This is the first study showing phenotypic selection for distinct CEACAM-binding phenotypes in vivo, and it supports the opposing functions of CEACAMs that facilitate infection versus driving inflammation within the genital tract. PMID:25605771

  5. A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

    PubMed Central

    Schwartz, Michael P.; Rogers, Robert E.; Singh, Samir P.; Lee, Justin Y.; Loveland, Samuel G.; Koepsel, Justin T.; Witze, Eric S.; Montanez-Sauri, Sara I.; Sung, Kyung E.; Tokuda, Emi Y.; Sharma, Yasha; Everhart, Lydia M.; Nguyen, Eric H.; Zaman, Muhammad H.; Beebe, David J.; Ahn, Natalie G.; Murphy, William L.; Anseth, Kristi S.

    2013-01-01

    Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results

  6. The oxygen consumption of the placenta and foetal membranes in the sheep

    PubMed Central

    Campbell, A. G. M.; Dawes, G. S.; Fishman, A. P.; Hyman, A. I.; James, G. B.

    1966-01-01

    1. In anaesthetized sheep near term the O2 uptake of the foetus from the placenta was reduced to zero either by replacing the foetus with a mechanical pump, or by ventilation of the foetus after delivery or in utero. When umbilical arterial and venous PO2 were made equal, they were much less than maternal arterial PO2. 2. This difference in PO2 was attributed to O2 consumption both within the placental cotyledons and the extra-cotyledonary foetal membranes. 3. The foetal membranes and cord in the sheep are supplied by branches of umbilical blood vessels and constitute an oxygen-consuming by-pass on the foetal side of the placenta. 4. The O2 consumption of individual placental cotyledons was measured in vivo. 5. The combined O2 consumption of the whole placenta and foetal membranes in vivo was a considerable fraction of foetal O2 consumption. 6. The consequences of these observations are discussed in relation to O2 transfer across the placenta. ImagesFig. 8Fig. 9 PMID:5949598

  7. Effect of Embryo Vitrification on Rabbit Foetal Placenta Proteome during Pregnancy

    PubMed Central

    Saenz-de-Juano, Maria Desemparats; Vicente, José Salvador; Hollung, Kristin; Marco-Jiménez, Francisco

    2015-01-01

    Very limited information on the post-implantatory effects of vitrification has been published till now. We observed in a previous study that the vitrification procedure for the cryopreservation of embryos introduced transcriptomic and proteomic modifications in the rabbit foetal placenta at the middle of gestation. Now, we have conducted a proteomic study to determine whether protein alterations in the foetal placenta induced by the vitrification procedure remain during pregnancy. In this study, we used 2D-DIGE and mass spectrometry (MALDI-TOF-TOF and LC-MS/MS analysis) to identify the protein changes during middle and late stages of gestation (Day 14 and Day 24, respectively) in rabbit foetal placenta. We identified 11 differentially expressed proteins at Day 14 and 13 proteins at Day 24. Data are available via ProteomeXchange with identifiers PXD001840 and PXD001836. In addition, we demonstrate the presence of three proteins, serum albumin, isocitrate dehydrogenase 1 [NADP+], and phosphoglycerate mutase 1, which were altered during pregnancy. We demonstrate the existence of changes in foetal placental protein during pregnancy induced by the vitrification procedure, which brings into question whether vitrification effects observed during foetal development could lead to physiological and metabolic disorders in adulthood. This effect, taken together with other effects reported in the literature, suggests that embryo cryopreservation is not neutral. PMID:25915775

  8. Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype

    PubMed Central

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft

  9. Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines

    SciTech Connect

    Gu Yongpeng; Li Hongzhen; Miki, Jun; Kim, Kee-Hong; Furusato, Bungo; Sesterhenn, Isabell A.; Chu, Wei-Sing; McLeod, David G.; Srivastava, Shiv; Ewing, Charles M.; Isaacs, William B.; Rhim, Johng S. . E-mail: jrhim@cpdr.org

    2006-04-01

    In vitro human prostate cell culture models are critical for clarifying the mechanism of prostate cancer progression and for testing preventive and therapeutic agents. Cell lines ideal for the study of human primary prostate tumors would be those derived from spontaneously immortalized tumor cells; unfortunately, explanted primary prostate cells survive only short-term in culture, and rarely immortalize spontaneously. Therefore, we recently have generated five immortal human prostate epithelial cell cultures derived from both the benign and malignant tissues of prostate cancer patients with telomerase, a gene that prevents cellular senescence. Examination of these cell lines for their morphologies and proliferative capacities, their abilities to grow in low serum, to respond to androgen stimulation, to grow above the agar layer, to form tumors in SCID mice, suggests that they may serve as valid, useful tools for the elucidation of early events in prostate tumorigenesis. Furthermore, the chromosome alterations observed in these immortalized cell lines expressing aspects of the malignant phenotypes imply that these cell lines accurately recapitulate the genetic composition of primary tumors. These novel in vitro models may offer unique models for the study of prostate carcinogenesis and also provide the means for testing both chemopreventive and chemotherapeutic agents.

  10. THE HUMAN GENE MAP FOR PERFORMANCE AND HEALTH-RELATED FITNESS PHENOTYPES: THE 2004 UPDATE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We began this series in 2000 with the aim of making available in an easily accessible format all the advances on the genetic basis of a large family of exercise-related traits. The current review presents the 2004 update of the human gene map for physical performance and health-related fitness pheno...

  11. Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression

    PubMed Central

    Park, Thomas I-H.; Feisst, Vaughan; Brooks, Anna E. S.; Rustenhoven, Justin; Monzo, Hector J.; Feng, Sheryl X.; Mee, Edward W.; Bergin, Peter S.; Oldfield, Robyn; Graham, E. Scott; Curtis, Maurice A.; Faull, Richard L. M.; Dunbar, P. Rod; Dragunow, Mike

    2016-01-01

    The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73+CD45− mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90+ cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90− population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation. PMID:27215737

  12. Phenotypic and genotypic characterization of Salmonella enterica serovar Dublin isolates from cattle and humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Antimicrobial resistance (AR), particularly multi-drug resistance (MDR), is of global concern. Salmonella causes approximately 93.8 million human infections worldwide/year including 1.2 million cases in the US. An increase in both AR and MDR among some serotypes has been observed. Sa...

  13. Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression.

    PubMed

    Park, Thomas I-H; Feisst, Vaughan; Brooks, Anna E S; Rustenhoven, Justin; Monzo, Hector J; Feng, Sheryl X; Mee, Edward W; Bergin, Peter S; Oldfield, Robyn; Graham, E Scott; Curtis, Maurice A; Faull, Richard L M; Dunbar, P Rod; Dragunow, Mike

    2016-01-01

    The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73(+)CD45(-) mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90(+) cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90(-) population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation. PMID:27215737

  14. Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype

    PubMed Central

    Kristiansen, G; Rose, M; Geisler, C; Fritzsche, F R; Gerhardt, J; Lüke, C; Ladhoff, A-M; Knüchel, R; Dietel, M; Moch, H; Varga, Z; Theurillat, J-P; Gorr, T A; Dahl, E

    2010-01-01

    Background: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. Methods: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-α (ERα)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERα or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. Results: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2α and CAIX, but not to HIF-1α or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERα or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. Conclusion: We conclude that in breast cancer, Mb is co-expressed with ERα and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours. PMID:20531416

  15. The phenotype and tissue-specific nature of multipotent cells derived from human mature adipocytes.

    PubMed

    Kou, Liang; Lu, Xiao-Wen; Wu, Min-Ke; Wang, Hang; Zhang, Yu-Jiao; Sato, Soh; Shen, Jie-Fei

    2014-02-21

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have been considered to be a homogeneous group of multipotent cells, which present to be an alternative source of adult stem cells for regenerative medicine. However, many aspects of the cellular nature about DFAT cells remained unclarified. This study aimed to elucidate the basic characteristics of DFAT cells underlying their functions and differentiation potentials. By modified ceiling culture technique, DFAT cells were converted from human mature adipocytes from the human buccal fat pads. Flow cytometry analysis revealed that those derived cells were a homogeneous population of CD13(+) CD29(+) CD105(+) CD44(+) CD31(-) CD34(-) CD309(-) α-SMA(-) cells. DFAT cells in this study demonstrated tissue-specific differentiation properties with strong adipogenic but much weaker osteogenic capacity. Neither did they express endothelial markers under angiogenic induction. PMID:24486314

  16. Human asthma phenotypes: from the clinic, to cytokines, and back again

    PubMed Central

    Bhakta, Nirav R.; Woodruff, Prescott G.

    2012-01-01

    Summary A large body of experimental evidence supports the hypothesis that T-helper 2 (Th2) cytokines orchestrate allergic airway inflammation in animal models. However, human asthma is heterogeneous with respect to clinical features, cellular sources of inflammation, and response to common therapies. This disease heterogeneity has been investigated using sputum cytology as well as unbiased clustering approaches using cellular and clinical data. Important differences in cytokine-driven inflammation may underlie this heterogeneity, and studies in human subjects with asthma have begun to elucidate these molecular differences. This molecular heterogeneity may be assessed by existing biomarkers (induced sputum evaluation or exhaled nitric oxide testing) or may require novel biomarkers. Effective testing and application of emerging therapies that target Th2 cytokines will depend on accurate and easily obtained biomarkers of this molecular heterogeneity in asthma. Furthermore, whether other non-Th2 cytokine pathways underlie airway inflammation in specific subsets of patients with asthma is an unresolved question and an important goal of future research using both mouse models and human studies. PMID:21682748

  17. Molecular basis of the attenuated phenotype of human APOBEC3B DNA mutator enzyme

    PubMed Central

    Caval, Vincent; Bouzidi, Mohamed S.; Suspène, Rodolphe; Laude, Hélène; Dumargne, Marie-Charlotte; Bashamboo, Anu; Krey, Thomas; Vartanian, Jean-Pierre; Wain-Hobson, Simon

    2015-01-01

    The human APOBEC3A and APOBEC3B genes (A3A and A3B) encode DNA mutator enzymes that deaminate cytidine and 5-methylcytidine residues in single-stranded DNA (ssDNA). They are important sources of mutations in many cancer genomes which show a preponderance of CG->TA transitions. Although both enzymes can hypermutate chromosomal DNA in an experimental setting, only A3A can induce double strand DNA breaks, even though the catalytic domains of A3B and A3A differ by only 9% at the protein level. Accordingly we sought the molecular basis underlying A3B attenuation through the generation of A3A-A3B chimeras and mutants. It transpires that the N-terminal domain facilitates A3B activity while a handful of substitutions in the catalytic C-terminal domain impacting ssDNA binding serve to attenuate A3B compared to A3A. Interestingly, functional attenuation is also observed for the rhesus monkey rhA3B enzyme compared to rhA3A indicating that this genotoxic dichotomy has been selected for and maintained for some 38 million years. Expression of all human ssDNA cytidine deaminase genes is absent in mature sperm indicating they contribute to somatic mutation and cancer but not human diversity. PMID:26384561

  18. Reversal of the human and murine multidrug-resistance phenotype with megestrol acetate.

    PubMed

    Wang, L; Yang, C P; Horwitz, S B; Trail, P A; Casazza, A M

    1994-01-01

    MA is an orally active PG derivative with an excellent safety profile that is used primarily for the treatment of carcinomas of the breast and endometrium. We investigated the potential application of MA as an MDR-reversal agent using cell culture and human tumor xenograft models. The reversing activity of MA in vitro was compared with that of PG and VER in two human MDR cell lines, the colon carcinoma HCT-116/VM46 and the breast carcinoma MCF-7/ADR, and in a murine cell line, J774.2. At concentrations as low as 3 microM, MA was capable of partially restoring sensitivity to Act D in the HCT-116/VM46 cells and sensitivity to DOX in the MCF-7/ADR cells. Although less effective than VER, MA was about 2.5 times more potent than PG in reversing MDR at equimolar concentrations. Increased accumulation of DOX in drug-resistant cells that were treated simultaneously with MA was observed by flow cytometry. In vivo, using established human colon and breast carcinoma xenografts implanted s.c. in athymic mice, the combined therapy with MA and DOX resulted in enhanced antitumor activity relative to that of DOX alone in the MDR sublines. These results suggest that MA may be a promising clinical MDR-reversing agent. PMID:8194172

  19. Human Cpr (Cell Cycle Progression Restoration) Genes Impart a Far(-) Phenotype on Yeast Cells

    PubMed Central

    Edwards, M. C.; Liegeois, N.; Horecka, J.; DePinho, R. A.; Sprague-Jr., G. F.; Tyers, M.; Elledge, S. J.

    1997-01-01

    Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G(1) arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G(1) arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles. PMID:9383053

  20. Primary Human Chondrocyte Extracellular Matrix Formation and Phenotype Maintenance using RGD-derivatized PEGDM Hydrogels Possessing a Continuous Gradient in Modulus

    PubMed Central

    Smith Callahan, Laura A.; Ganios, Anna M.; Childers, Erin P.; Weiner, Scott D.; Becker, Matthew L.

    2013-01-01

    Efficient ex vivo methods for expanding primary human chondrocytes while maintaining phenotype is critical to advancing autologous cell sourcing efforts for tissue engineering applications. While there is significant activity in the literature, systematic approaches are necessary to determine and optimize the chemical and mechanical scaffold properties for hyaline cartilage generation using limited cell numbers. Functionalized hydrogels possessing continuous variations in physico-chemical properties are therefore an efficient three-dimensional platform for studying several properties simultaneously. Herein, we describe a polyethylene glycol dimethacrylate (PEGDM) hydrogel system possessing a gradient in modulus (~27,000 Pa to 3,800 Pa) containing a uniform concentration of arginine–glycine–aspartic acid peptide (RGD) to enhance cellular adhesion for the correlation of primary human osteoarthritic chondrocyte proliferation, phenotype maintenance, and ECM production to the hydrogel properties. Cell number and chondrogenic phenotype (CD14:CD90 ratios) were found to decline in regions with higher storage modulus (>13,100 Pa), while regions with lower storage modulus maintained cell number and phenotype. Over three weeks of culture, hydrogel regions possessing lower storage modulus experience an increase in ECM content (~200%) compared to regions with higher storage modulus. Variations in the amount and organization of cytoskeletal markers actin and vinculin were observed within the modulus gradient which are indicative of the differences in chondrogenic phenotype maintenance and ECM expression. Thus scaffold mechanical properties significantly impact on modulating human osteoarthritic chondrocyte behavior and tissue formation. PMID:23291491

  1. A novel low-profile ventriculoamniotic shunt for foetal aqueductal stenosis.

    PubMed

    Chen, Yanfei; Emery, Stephen P; Maxey, Antonina P; Gu, Xinzhu; Wagner, William R; Chun, Youngjae

    2016-01-01

    This study proposed a novel ventriculoamniotic shunt device for foetal aqueductal stenosis treatment fabricated with 3Fr or 4Fr size catheters that have a longitudinal bending stiffness with kink resistance, sufficient luminal area for cerebrospinal fluid drainage and capacity for valve integration. Computational flow dynamics studies were carried out to optimise the device design, including size of the lumen and length of the device. An in vitro pressure and flow rate measurement test circuit was constructed to assess the high pressure relieving functionality of draining cerebrospinal fluid from foetal brain. Additionally, a resistance force measurement test platform was built to quantitatively evaluate the anchor performance of various geometric designs. The valve functionality was qualitatively evaluated through the visualisation of the flow patterns in the amniotic sac with injected red coloured fluid under stereomicroscopy. These in vitro results demonstrate the feasibility of the ventriculoamniotic shunt device designed for placement in the foetal brain. PMID:27004923

  2. Sex Differences in the Association between Foetal Growth and Child Attention at Age Four: Specific Vulnerability of Girls

    ERIC Educational Resources Information Center

    Murray, Elizabeth; Matijasevich, Alicia; Santos, Iná S.; Barros, Aluísio J. D.; Anselmi, Luciana; Barros, Fernando C.; Stein, Alan

    2015-01-01

    Background: Recent evidence suggests that impaired foetal growth may provide an early indication of increased risk of child attention problems. However, despite both foetal growth and child attention problems differing by sex, few studies have examined sex differences in this association. Furthermore, no studies have been conducted in low- and…

  3. Short-term mechanical stretch fails to differentiate human adipose-derived stem cells into cardiovascular cell phenotypes

    PubMed Central

    2014-01-01

    Background We and others have previously demonstrated that adipose-derived stem cells (ASCs) transplantation improve cardiac dysfunction post-myocardium infarction (MI) under hemodynamic stress in rats. The beneficial effects appear to be associated with pleiotropic factors due to a complex interplay between the transplanted ASCs and the microenvironment in the absence of cell transdifferentiation. In the present work, we tested the hypothesis that mechanical stretch per se could change human ASCs (hASCs) into cardiovascular cell phenotypes that might influence post-MI outcomes. Methods Human ASCs were obtained from patients undergoing liposuction procedures. These cells were stretched 12%, 1Hz up to 96 hours by using Flexercell 4000 system. Protein and gene expression were evaluated to identify cardiovascular cell markers. Culture medium was analyzed to determine cell releasing factors, and contraction potential was also evaluated. Results Mechanical stretch, which is associated with extracellular signal-regulated kinase (ERK) phosphorylation, failed to induce the expression of cardiovascular cell markers in human ASCs, and mesenchymal cell surface markers (CD29; CD90) remained unchanged. hASCs and smooth muscle cells (SMCs) displayed comparable contraction ability. In addition, these cells demonstrated a profound ability to secrete an array of cytokines. These two properties of human ASCs were not influenced by mechanical stretch. Conclusions Altogether, our findings demonstrate that hASCs secrete an array of cytokines and display contraction ability even in the absence of induction of cardiovascular cell markers or the loss of mesenchymal surface markers when exposed to mechanical stretch. These properties may contribute to beneficial post-MI cardiovascular outcomes and deserve to be further explored under the controlled influence of other microenvironment components associated with myocardial infarction, such as tissue hypoxia. PMID:24885410

  4. Identification of metabolites of dalfampridine (4-aminopyridine) in human subjects and reaction phenotyping of relevant cytochrome P450 pathways

    PubMed Central

    Caggiano, Anthony; Blight, Andrew

    2013-01-01

    Objectives An extended release formulation of dalfampridine (4-aminopyridine; 4-AP), a potassium channel blocker is available in the USA to improve walking in patients with multiple sclerosis. This study investigated the human metabolites of 4-AP and the cytochrome P450 (CYP450) pathways responsible for 4-AP metabolism. Methods Metabolites were identified, using thin layer chromatography, high performance liquid chromatography, and gas chromatography/mass spectroscopy, in plasma and urine samples obtained during an excretion balance study of four subjects who were administered a single oral 15-mg dose of 14C-4-AP. Samples were compared with authentic standards of 4-AP, 2-hydroxy-4AP, 3-hydroxy-4AP, and 4-AP-N-oxide. Reaction phenotyping was performed in vitro using human liver microsomes and recombinant CYP450 enzymes with incubation in the presence of direct and time-dependent inhibitors to determine the CYP450 pathways involved in metabolite formation. Results While most (∼70%) of the radioactivity detected in plasma at each time point corresponded to unchanged 4-AP, two major metabolites were recovered. One metabolite co-localized with the authentic reference standard of 3-hydroxy-4-AP, and the other metabolite was identified as the sulfate conjugate of 3-hydroxy-4-AP. Two minor components were observed, one accounting for 2% of radioactivity and the other below the level of quantitation. Reaction phenotyping showed moderate correlations for conversion of 4-AP to 3-hydroxy-4AP with both CYP2E1 (r = 0.596; p < 0.001) and CYP2C8 (r = 0.608; p < 0.001). Use of a CYP2E1 metabolism-dependent inhibitor inhibited formation of 3-hydroxy-4-AP with and without pre-incubation (higher inhibition with pre-incubation), further supporting the likelihood of CYP2E1 as a metabolic pathway. The main limitation of this study was the inability to identify the CYP enzymes responsible for the 3-hydroxylation of 4-AP, although this conversion represents only a minor

  5. Azelaic acid reduced senescence-like phenotype in photo-irradiated human dermal fibroblasts: possible implication of PPARγ.

    PubMed

    Briganti, Stefania; Flori, Enrica; Mastrofrancesco, Arianna; Kovacs, Daniela; Camera, Emanuela; Ludovici, Matteo; Cardinali, Giorgia; Picardo, Mauro

    2013-01-01

    Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long-time treatment with AzA. We previously unrevealed that anti-inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo-aggravated disease, we investigated the ability of AzA to counteract stress-induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8-methoxypsoralen (PUVA), previously reported to activate a senescence-like phenotype, including long-term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence-associated β-galactosidase (SA-β-gal). We found that PUVA-treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP-1 release and SA-β-galactosidase-positive cells. Moreover, AzA induced a reduction in ROS generation, an up-modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA-treated HDFs. Further evidences of AzA anti-senescence effect were repression of p53 and p21, increase in type I pro-collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA-SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA-induced senescence-like phenotype and its ability to activate PPAR-γ provides relevant insights into the anti-senescence mechanism. PMID:23278893

  6. Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I

    PubMed Central

    Li, Kairong; Turner, Ashley N.; Chen, Min; Brosius, Stephanie N.; Schoeb, Trenton R.; Messiaen, Ludwine M.; Bedwell, David M.; Zinn, Kurt R.; Anastasaki, Corina; Gutmann, David H.; Korf, Bruce R.

    2016-01-01

    ABSTRACT Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1Arg681* and missense NF1Gly848Arg mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1Gly848Arg mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1Arg681* mutation are not viable. Mice with one Nf1Arg681* allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf14F/Arg681*; DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1. PMID:27482814

  7. TAK1 is a key modulator of the profibrogenic phenotype of human ileal myofibroblasts in Crohn's disease.

    PubMed

    Grillo, Alessia Rosaria; Scarpa, Melania; D'Incà, Renata; Brun, Paola; Scarpa, Marco; Porzionato, Andrea; De Caro, Raffaele; Martines, Diego; Buda, Andrea; Angriman, Imerio; Palù, Giorgio; Sturniolo, Giacomo Carlo; Castagliuolo, Ignazio

    2015-09-15

    Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) signaling can mediate inflammatory responses as well as tissue remodeling. Intestinal mucosal myofibroblast (IMF) activation drives gut fibrosis in Crohn's disease (CD); however, the molecular pathways involved are largely unknown. Thus we investigated the yet-unknown expression and function of TAK1 in human CD-associated fibrosis. Ileal surgical specimens, ileal biopsies, and IMF isolated from controls and CD patients were analyzed for TAK1 and its active phosphorylated form (pTAK1) by Western blotting, immunohistochemistry, and real-time quantitative PCR. TAK1 pharmacological inhibition and silencing were used to assess its role in collagen and inflammatory cytokine synthesis in IMF. TAK1 and pTAK1 levels increased in ileum specimens from CD patients compared with controls and correlated to tissue fibrosis. Similarly, TAK1 mRNA in ileal biopsies of CD patients correlated with fibrogenic marker expression but not inflammatory cytokines. CD-derived IMF showed higher TAK1 and pTAK1 expression associated with increased collagen1(α)1 mRNA levels compared with control IMF. TGF-β1 promoted pTAK1 nuclear translocation and collagen synthesis. TAK1 inhibition or silencing significantly reduced TGF-β1-stimulated collagen production and normalized the profibrogenic phenotype of CD-derived IMF. Taken together, these data suggest that TAK1 activation and nuclear translocation induce and maintain a fibrogenic phenotype in the IMF. Thus the TAK1 signaling pathway may represent a suitable target to design new, antifibrotic therapies. PMID:26185333

  8. Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I.

    PubMed

    Li, Kairong; Turner, Ashley N; Chen, Min; Brosius, Stephanie N; Schoeb, Trenton R; Messiaen, Ludwine M; Bedwell, David M; Zinn, Kurt R; Anastasaki, Corina; Gutmann, David H; Korf, Bruce R; Kesterson, Robert A

    2016-07-01

    Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by the occurrence of nerve sheath tumors and considerable clinical heterogeneity. Some translational studies have been limited by the lack of animal models available for assessing patient-specific mutations. In order to test therapeutic approaches that might restore function to the mutated gene or gene product, we developed mice harboring NF1 patient-specific mutations including a nonsense mutation (c.2041C>T; p.Arg681*) and a missense mutation (c.2542G>C; p.Gly848Arg). The latter is associated with the development of multiple plexiform neurofibromas along spinal nerve roots. We demonstrate that the human nonsense NF1(Arg681*) and missense NF1(Gly848Arg) mutations have different effects on neurofibromin expression in the mouse and each recapitulates unique aspects of the NF1 phenotype, depending upon the genetic context when assessed in the homozygous state or when paired with a conditional knockout allele. Whereas the missense Nf1(Gly848Arg) mutation fails to produce an overt phenotype in the mouse, animals homozygous for the nonsense Nf1(Arg681*) mutation are not viable. Mice with one Nf1(Arg681*) allele in combination with a conditional floxed Nf1 allele and the DhhCre transgene (Nf1(4F/Arg681*); DhhCre) display disorganized nonmyelinating axons and neurofibromas along the spinal column, which leads to compression of the spinal cord and paralysis. This model will be valuable for preclinical testing of novel nonsense suppression therapies using drugs to target in-frame point mutations that create premature termination codons in individuals with NF1. PMID:27482814

  9. A position effect on TRPS1 is associated with Ambras syndrome in humans and the Koala phenotype in mice

    PubMed Central

    Fantauzzo, Katherine A.; Tadin-Strapps, Marija; You, Yun; Mentzer, Sarah E.; Baumeister, Friedrich A.M.; Cianfarani, Stefano; Van Maldergem, Lionel; Warburton, Dorothy; Sundberg, John P.; Christiano, Angela M.

    2008-01-01

    Ambras syndrome (AS) is a rare form of congenital hypertrichosis with excessive hair on the shoulders, face and ears. Cytogenetic studies have previously implicated an association with rearrangements of chromosome 8. Here we define an 11.5 Mb candidate interval for AS on chromosome 8q based on cytogenetic breakpoints in three patients. TRPS1, a gene within this interval, was deleted in a patient with an 8q23 chromosomal rearrangement, while its expression was significantly downregulated in another patient with an inversion breakpoint 7.3 Mb downstream of TRPS1. Here, we describe the first potential long-range position effect on the expression of TRPS1. To gain insight into the mechanisms by which Trps1 affects the hair follicle, we performed a detailed analysis of the hair abnormalities in Koa mice, a mouse model of hypertrichosis. We found that the proximal breakpoint of the Koa inversion is located 791 kb upstream of Trps1. Quantitative real-time polymerase chain reaction, in situ hybridization and immunofluorescence analysis revealed that Trps1 expression levels are reduced in Koa mutant mice at the sites of pathology for the phenotype. We determined that the Koa inversion creates a new Sp1 binding site and translocates additional Sp1 binding sites within a highly conserved stretch spanning the proximal breakpoint, providing a potential mechanism for the position effect. Collectively, these results describe a position effect that downregulates TRPS1 expression as the probable cause of hypertrichosis in AS in humans and the Koa phenotype in mice. PMID:18713754

  10. Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization.

    PubMed Central

    Lenz, A; Heine, M; Schuler, G; Romani, N

    1993-01-01

    Dendritic cells (DC) comprise a system of cells in lymphoid and nonlymphoid organs that are specialized to present antigens and to initiate primary T cell responses. The Langerhans cell of the epidermis is used as a prototype for studies of DC in the skin. We have characterized a population of DC in human dermis, one of the first examples of these cells in nonlymphoid organs other than epidermis. To identify their distinct functions and phenotype, we relied upon the preparation of enriched populations that emigrate from organ explants of dermis. The dermal cells have the following key features of mature DC: (a) sheet-like processes, or veils, that are constantly moving; (b) very high levels of surface MHC products; (c) absence of markers for macrophages, lymphocytes, and endothelium; (d) substantial expression of adhesion/costimulatory molecules such as CD11/CD18, CD54 (ICAM-1), B7/BB1, CD40; and (e) powerful stimulatory function for resting T cells. Dermal DC are fully comparable to epidermis-derived DC, except for the lack of Birbeck granules, lower levels of CD1a, and higher levels of CD36. DC were also detected in explants of mouse dermis. We conclude that cutaneous DC include both epidermal and dermal components, and suggest that other human nonlymphoid tissues may also serve as sources of typical immunostimulatory DC. Images PMID:8254016

  11. Elucidating molecular phenotypes caused by the SORL1 Alzheimer's disease genetic risk factor using human induced pluripotent stem cells.

    PubMed

    Young, Jessica E; Boulanger-Weill, Jonathan; Williams, Daniel A; Woodruff, Grace; Buen, Floyd; Revilla, Arra C; Herrera, Cheryl; Israel, Mason A; Yuan, Shauna H; Edland, Steven D; Goldstein, Lawrence S B

    2015-04-01

    Predisposition to sporadic Alzheimer's disease (SAD) involves interactions between a person's unique combination of genetic variants and the environment. The molecular effect of these variants may be subtle and difficult to analyze with standard in vitro or in vivo models. Here we used hIPSCs to examine genetic variation in the SORL1 gene and possible contributions to SAD-related phenotypes in human neurons. We found that human neurons carrying SORL1 variants associated with an increased SAD risk show a reduced response to treatment with BDNF, at the level of both SORL1 expression and APP processing. shRNA knockdown of SORL1 demonstrates that the differences in BDNF-induced APP processing between genotypes are dependent on SORL1 expression. We propose that the variation in SORL1 expression induction by BDNF is modulated by common genetic variants and can explain how genetic variation in this one locus can contribute to an individual's risk of developing SAD. PMID:25772071

  12. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry.

    PubMed

    Leelatian, Nalin; Diggins, Kirsten E; Irish, Jonathan M

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25-35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank ( http://www.cytobank.org/irishlab/ ). PMID:26542718

  13. Characterizing Phenotypes and Signaling Networks of Single Human Cells by Mass Cytometry

    PubMed Central

    Leelatian, Nalin; Diggins, Kirsten E.; Irish, Jonathan M.

    2015-01-01

    Single cell mass cytometry is revolutionizing our ability to quantitatively characterize cellular biomarkers and signaling networks. Mass cytometry experiments routinely measure 25–35 features of each cell in primary human tissue samples. The relative ease with which a novice user can generate a large amount of high quality data and the novelty of the approach have created a need for example protocols, analysis strategies, and datasets. In this chapter, we present detailed protocols for two mass cytometry experiments designed as training tools. The first protocol describes detection of 26 features on the surface of human peripheral blood mononuclear cells. In the second protocol, a mass cytometry signaling network profile measures 25 node states comprised of five key signaling effectors (AKT, ERK1/2, STAT1, STAT5, and p38) quantified under five conditions (Basal, FLT3L, SCF, IL-3, and IFNγ). This chapter compares manual and unsupervised data analysis approaches, including bivariate plots, heatmaps, histogram overlays, SPADE, and viSNE. Data files in this chapter have been shared online using Cytobank (http://www.cytobank.org/irishlab/). PMID:26542718

  14. Phenomics Research on Coronary Heart Disease Based on Human Phenotype Ontology

    PubMed Central

    Shi, Qi; Gao, Kuo; Zhao, Huihui; Wang, Juan; Zhai, Xing; Chen, Jianxin; Wang, Wei

    2014-01-01

    The characteristics of holistic, dynamics, complexity, and spatial and temporal features enable “Omics” and theories of TCM to interlink with each other. HPO, namely, “characterization,” can be understood as a sorting and generalization of the manifestations shown by people with diseases on the basis of the phenomics. Syndrome is the overall “manifestation” of human body pathological and physiological changes expressed by four diagnostic methods' information. The four diagnostic methods' data could be the most objective and direct manifestations of human body under morbid conditions. In this aspect, it is consistent with the connation of “characterization.” Meanwhile, the four diagnostic methods' data also equip us with features of characterization in HPO. In our study, we compared 107 pieces of four diagnostic methods' information with the “characterization database” to further analyze data of four diagnostic methods' characterization in accordance with the common characteristics of four diagnostic methods' information and characterization and integrated 107 pieces of four diagnostic methods' data to relevant items in HPO and finished the expansion of characterization information in HPO. PMID:25610858

  15. Overexpression of human loricrin in transgenic mice produces a normal phenotype.

    PubMed Central

    Yoneda, K; Steinert, P M

    1993-01-01

    The cornified cell envelope (CE) of terminally differentiating stratified squamous epithelial cells is a complex multiprotein assembly about 15 nm thick of which loricrin is a major component. We have produced transgenic mice bearing the human loricrin transgene in order to study the role of loricrin in CE assembly, structure, and function. By analyses of RNA and protein, we show that the human transgene is expressed in mouse epithelial tissues in an appropriate developmental manner but at an overall level about twice that of endogenous mouse loricrin. Thus the 20-kbp construct used contains all necessary regulatory elements. By immunogold electron microscopy, all of the expressed protein is incorporated into the CE. That no alternations were noted indicates that overproduction of the loricrin component of the CE does not affect the flexible structure or function of the epithelial tissues. Furthermore, these data imply that loricrin may be the last protein to be deposited onto, and thus lines, the intracellular surface of the CE, where it may be accessible to interact with the subjacent keratin intermediate-filament network. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8248167

  16. PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype

    PubMed Central

    Duan, Shunlei; Yuan, Guohong; Liu, Xiaomeng; Ren, Ruotong; Li, Jingyi; Zhang, Weizhou; Wu, Jun; Xu, Xiuling; Fu, Lina; Li, Ying; Yang, Jiping; Zhang, Weiqi; Bai, Ruijun; Yi, Fei; Suzuki, Keiichiro; Gao, Hua; Esteban, Concepcion Rodriguez; Zhang, Chuanbao; Belmonte, Juan Carlos Izpisua; Chen, Zhiguo; Wang, Xiaomin; Jiang, Tao; Qu, Jing; Tang, Fuchou; Liu, Guang-Hui

    2015-01-01

    PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs), but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs, binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7, which in turn promotes oncogenic transformation of NSCs and instates ‘aggressiveness' in human glioblastoma stem cells. In a large clinical database, we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore, we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together, we uncover a potential mechanism of how PTEN safeguards NSCs, and establish a cellular platform to identify factors involved in NSC transformation, potentially permitting personalized treatment of glioblastoma. PMID:26632666

  17. Evaluation of Phenotypic and Genotypic Methods for Subtyping Campylobacter jejuni Isolates from Humans, Poultry, and Cattle

    PubMed Central

    Nielsen, Eva Møller; Engberg, Jørgen; Fussing, Vivian; Petersen, Lise; Brogren, Carl-Henrik; On, Stephen L. W.

    2000-01-01

    Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods. PMID:11015406

  18. Interactions between HERC2, OCA2 and MC1R may influence human pigmentation phenotype.

    PubMed

    Branicki, Wojciech; Brudnik, Urszula; Wojas-Pelc, Anna

    2009-03-01

    Human pigmentation is a polygenic trait which may be shaped by different kinds of gene-gene interactions. Recent studies have revealed that interactive effects between HERC2 and OCA2 may be responsible for blue eye colour determination in humans. Here we performed a population association study, examining important polymorphisms within the HERC2 and OCA2 genes. Furthermore, pooling these results with genotyping data for MC1R, ASIP and SLC45A2 obtained for the same population sample we also analysed potential genetic interactions affecting variation in eye, hair and skin colour. Our results confirmed the association of HERC2 rs12913832 with eye colour and showed that this SNP is also significantly associated with skin and hair colouration. It is also concluded that OCA2 rs1800407 is independently associated with eye colour. Finally, using various approaches we were able to show that there is an interaction between MC1R and HERC2 in determination of skin and hair colour in the studied population sample. PMID:19208107

  19. Decreasing matrix modulus of PEG hydrogels induces a vascular phenotype in human cord blood stem cells.

    PubMed

    Mahadevaiah, Shruthi; Robinson, Karyn G; Kharkar, Prathamesh M; Kiick, Kristi L; Akins, Robert E

    2015-09-01

    Adult and congenital cardiovascular diseases are significant health problems that are often managed using surgery. Bypass grafting is a principal therapy, but grafts fail at high rates due to hyperplasia, fibrosis, and atherosclerosis. Biocompatible, cellularized materials that attenuate these complications and encourage healthy microvascularization could reduce graft failure, but an improved understanding of biomaterial effects on human stem cells is needed to reach clinical utility. Our group investigates stem-cell-loaded biomaterials for placement along the adventitia of at-risk vessels and grafts. Here, the effects of substrate modulus on human CD34+ stem cells from umbilical cord blood were evaluated. Cells were isolated by immunomagnetic separation and encapsulated in 3, 4, and 6 weight% PEG hydrogels containing 0.032% gelatin and 0.0044% fibronectin. Gels reached moduli of 0.34, 4.5, and 9.1 kPa. Cell viability approached 100%. Cell morphologies appeared similar across gels, but proliferation was significantly lower in 6 wt% gels. Expression profiling using stem cell signaling arrays indicated enhanced self-renewal and differentiation into vascular endothelium among cells in the lower weight percent gels. Thus, modulus was associated with cell proliferation and function. Gels with moduli in the low kilopascal range may be useful in stimulating cell engraftment and microvascularization of graft adventitia. PMID:26016692

  20. Long-term neural and physiological phenotyping of a single human

    PubMed Central

    Poldrack, Russell A.; Laumann, Timothy O.; Koyejo, Oluwasanmi; Gregory, Brenda; Hover, Ashleigh; Chen, Mei-Yen; Gorgolewski, Krzysztof J.; Luci, Jeffrey; Joo, Sung Jun; Boyd, Ryan L.; Hunicke-Smith, Scott; Simpson, Zack Booth; Caven, Thomas; Sochat, Vanessa; Shine, James M.; Gordon, Evan; Snyder, Abraham Z.; Adeyemo, Babatunde; Petersen, Steven E.; Glahn, David C.; Reese Mckay, D.; Curran, Joanne E.; Göring, Harald H. H.; Carless, Melanie A.; Blangero, John; Dougherty, Robert; Leemans, Alexander; Handwerker, Daniel A.; Frick, Laurie; Marcotte, Edward M.; Mumford, Jeanette A.

    2015-01-01

    Psychiatric disorders are characterized by major fluctuations in psychological function over the course of weeks and months, but the dynamic characteristics of brain function over this timescale in healthy individuals are unknown. Here, as a proof of concept to address this question, we present the MyConnectome project. An intensive phenome-wide assessment of a single human was performed over a period of 18 months, including functional and structural brain connectivity using magnetic resonance imaging, psychological function and physical health, gene expression and metabolomics. A reproducible analysis workflow is provided, along with open access to the data and an online browser for results. We demonstrate dynamic changes in brain connectivity over the timescales of days to months, and relations between brain connectivity, gene expression and metabolites. This resource can serve as a testbed to study the joint dynamics of human brain and metabolic function over time, an approach that is critical for the development of precision medicine strategies for brain disorders. PMID:26648521

  1. Demographic and Phenotypic Effects of Human Mediated Trophic Subsidy on a Large Australian Lizard (Varanus varius): Meal Ticket or Last Supper?

    PubMed Central

    Jessop, Tim S.; Smissen, Peter; Scheelings, Franciscus; Dempster, Tim

    2012-01-01

    Humans are increasingly subsidizing and altering natural food webs via changes to nutrient cycling and productivity. Where human trophic subsidies are concentrated and persistent within natural environments, their consumption could have complex consequences for wild animals through altering habitat preferences, phenotypes and fitness attributes that influence population dynamics. Human trophic subsidies conceptually create both costs and benefits for animals that receive increased calorific and altered nutritional inputs. Here, we evaluated the effects of a common terrestrial human trophic subsidies, human food refuse, on population and phenotypic (comprising morphological and physiological health indices) parameters of a large predatory lizard (∼2 m length), the lace monitor (Varanus varius), in southern Australia by comparison with individuals not receiving human trophic subsidies. At human trophic subsidies sites, lizards were significantly more abundant and their sex ratio highly male biased compared to control sites in natural forest. Human trophic subsidies recipient lizards were significantly longer, heavier and in much greater body condition. Blood parasites were significantly lower in human trophic subsidies lizards. Collectively, our results imply that human trophic subsidized sites were especially attractive to adult male lace monitors and had large phenotypic effects. However, we cannot rule out that the male-biased aggregations of large monitors at human trophic subsidized sites could lead to reductions in reproductive fitness, through mate competition and offspring survival, and through greater exposure of eggs and juveniles to predation. These possibilities could have negative population consequences. Aggregations of these large predators may also have flow on effects to surrounding food web dynamics through elevated predation levels. Given that flux of energy and nutrients into food webs is central to the regulation of populations and their

  2. A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells

    PubMed Central

    Vert, Anna; Castro, Jessica; Ribó, Marc; Benito, Antoni; Vilanova, Maria

    2016-01-01

    Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells. PMID:26918450

  3. A nuclear-directed human pancreatic ribonuclease (PE5) targets the metabolic phenotype of cancer cells.

    PubMed

    Vert, Anna; Castro, Jessica; Ribó, Marc; Benito, Antoni; Vilanova, Maria

    2016-04-01

    Ribonucleases represent a new class of antitumor RNA-damaging drugs. However, many wild-type members of the vertebrate secreted ribonuclease family are not cytotoxic because they are not able to evade the cytosolic ribonuclease inhibitor. We previously engineered the human pancreatic ribonuclease to direct it to the cell nucleus where the inhibitor is not present. The best characterized variant is PE5 that kills cancer cells through apoptosis mediated by the p21WAF1/CIP1 induction and the inactivation of JNK. Here, we have used microarray-derived transcriptional profiling to identify PE5 regulated genes on the NCI/ADR-RES ovarian cancer cell line. RT-qPCR analyses have confirmed the expression microarray findings. The results show that PE5 cause pleiotropic effects. Among them, it is remarkable the down-regulation of multiple genes that code for enzymes involved in deregulated metabolic pathways in cancer cells. PMID:26918450

  4. Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase

    SciTech Connect

    Nogueira, C.P.; McGuire, M.C.; Adkins, S.; Van Der Spek, A.F.L.; La Du, B.N. ); Bartels, C.F.; Lockridge, O. Eppley Institute, Univ. of Nebraska Medical Center, Omaha, NE ); Lubrano, T.; Rubinstein, H.M. Loyola Univ. Stritch School of Medicine, Maywood, IL ); Lightstone, H. )

    1992-10-01

    The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier the authors reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work they have identified two different point mutations associated with the fluoride-resistant phentotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members. 36 refs., 8 figs.

  5. Knock-in of human BACE1 cleaves murine APP and reiterates Alzheimer-like phenotypes.

    PubMed

    Plucińska, Kaja; Crouch, Barry; Koss, David; Robinson, Lianne; Siebrecht, Michael; Riedel, Gernot; Platt, Bettina

    2014-08-01

    Key neuropathological hallmarks of Alzheimer's disease (AD) are elevated levels of amyloid β-peptide (Aβ) species generated via amyloid precursor protein (APP) endoproteolysis and cleavage by the rate-limiting β-site enzyme 1 (BACE1). Because rodents do not develop amyloid pathologies, we here investigated whether AD-like endophenotypes can be created in mice by expression of human bace1. To avoid pitfalls of existing models, we introduced hbace1 via knock-in under the control of the CaMKII α promoter into the safe HPRT locus. We report amyloidogenic processing of murine APP in the hBACE1 mice (termed PLB4), resulting in the formation of toxic APP metabolites that accumulate intra- and extraneuronally in hippocampus and cortex. Pronounced accumulation of Aβ*56 and Aβ hexamers in the absence of plaque deposition was detected in brain tissue from symptomatic PLB4 mice. Heightened levels of inflammation (gliosis) also appeared in several AD-related brain regions (dentate gyrus, hippocampal area CA1, piriform and parietal cortices) at 6 and 12 months of age. Behaviorally, deficits in habituation to a novel environment and semantic-like memory (social transmission of food preference) were detected from 3 to 4 months of age. Impairments in spatial learning strategies in long-term reference (water maze) and working memory (Y-maze) tasks presented at 6 months, and were distinct from reductions in locomotor activity and anxiety. Overall, our data indicate for the first time that targeted, subtle forebrain-specific expression through single gene knock-in of hBACE1 is sufficient to generate AD-relevant cognitive impairments amid corresponding histopathologies, confirming human BACE as the key parameter in amyloid pathogenesis. PMID:25100603

  6. PAF-receptor is preferentially expressed in a distinct synthetic phenotype of smooth muscle cells cloned from human internal thoracic artery: Functional implications in cell migration

    SciTech Connect

    Stengel, Dominique; O'Neil, Caroline; Brocheriou, Isabelle; Karabina, Sonia-Athina; Durand, Herve; Caplice, Noel M.; Pickering, J. Geoffrey; Ninio, Ewa . E-mail: ninio@chups.jussieu.fr

    2006-08-04

    Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation.

  7. Human antimicrobial protein hCAP18/LL-37 promotes a metastatic phenotype in breast cancer

    PubMed Central

    Weber, Günther; Chamorro, Clara Ibel; Granath, Fredrik; Liljegren, Annelie; Zreika, Sami; Saidak, Zuzana; Sandstedt, Bengt; Rotstein, Samuel; Mentaverri, Romuald; Sánchez, Fabio; Pivarcsi, Andor; Ståhle, Mona

    2009-01-01

    Introduction Human cathelicidin antimicrobial protein, hCAP18, and its C-terminal peptide LL-37 is a multifunctional protein. In addition to being important in antimicrobial defense, it induces chemotaxis, stimulates angiogenesis and promotes tissue repair. We previously showed that human breast cancer cells express high amounts of hCAP18, and hypothesised that hCAP18/LL-37 may be involved in tumour progression. Methods hCAP18 mRNA was quantified in 109 primary breast cancers and compared with clinical findings and ERBB2 mRNA expression. Effects of exogenous LL-37 and transgenic overexpression of hCAP18 on ErbB2 signalling were investigated by immunoblotting using extracts from breast cancer cell lines ZR75-1 and derivatives of MCF7. We further analysed the impact of hCAP18/LL-37 on the morphology of breast cancer cells grown in soft agar, on cell migration and on tumour development in severe combined immunodeficiency (SCID) mice. Results The expression of hCAP18 correlated closely with that of ERBB2 and with the presence of lymph node metastases in oestrogen receptor-positive tumours. hCAP18/LL-37 amplified Heregulin-induced mitogen-activated protein kinase (MAPK) signalling through ErbB2, identifying a functional association between hCAP18/LL-37 and ErbB2 in breast cancer. Treatment with LL-37 peptide significantly stimulated the migration of breast cancer cells and their colonies acquired a dispersed morphology indicative of increased metastatic potential. A truncated version of LL-37 competitively inhibited LL-37 induced MAPK phosphorylation and significantly reduced the number of altered cancer cell colonies induced by LL-37 as well as suppressed their migration. Transgenic overexpression of hCAP18 in a low malignant breast cancer cell line promoted the development of metastases in SCID mice, and analysis of hCAP18 transgenic tumours showed enhanced activation of MAPK signalling. Conclusions Our results provide evidence that hCAP18/LL-37 contributes to breast

  8. PLZF Regulates CCR6 and is Critical for the Acquisition and Maintenance of the Th17 Phenotype in Human Cells

    PubMed Central

    Singh, Satya P.; Zhang, Hongwei H.; Tsang, Hsinyi; Gardina, Paul J.; Myers, Timothy G.; Nagarajan, Vijayaraj; Lee, Chang Hoon; Farber, Joshua M.

    2015-01-01

    Th17 cells, which all express the chemokine receptor CCR6, are implicated in many immune-mediated disorders such as psoriasis and multiple sclerosis. We found that expression levels of CCR6 on human effector/memory CD4+ T cells reflect a continuum of Th17 differentiation. By evaluating the transcriptome in cells with increasing CCR6, we detected progressive up-regulation of ZBTB16, which encodes the BTB-ZF transcription factor PLZF. Using ChIP for modified histones, p300, and PLZF, we identified enhancer-like sites at −9/−10 kb and −13/−14 kb from the upstream transcription start site of CCR6 that bind PLZF in CCR6+ cells. For Th cells from adult blood, both in the CCR6+ memory population and in naïve cells activated ex vivo, knockdown of ZBTB16 down-regulated CCR6 and other Th17-associated genes. ZBTB16 and RORC (which encodes the “master regulator” RORγt) cross-regulate each other, and PLZF binds at the RORC promoter in CCR6+ cells. In naive Th cells from cord blood, ZBTB16 expression was confined to the CD161+ cells, which are the Th17 cell precursors. ZBTB16 was not expressed in mouse Th17 cells and Th17 cells could be made from luxoid mice, which harbor an inactivating mutation in Zbtb16. These studies demonstrate a role for PLZF as an activator of transcription important both for Th17 differentiation and the maintenance of the Th17 phenotype in human cells, expand the role of PLZF as a critical regulator in the human adaptive immune system, and identify a novel, essential element in a regulatory network that is of significant therapeutic interest. PMID:25833398

  9. High mitochondrial respiration and glycolytic capacity represent a metabolic phenotype of human tolerogenic dendritic cells.

    PubMed

    Malinarich, Frano; Duan, Kaibo; Hamid, Raudhah Abdull; Bijin, Au; Lin, Wu Xue; Poidinger, Michael; Fairhurst, Anna-Marie; Connolly, John E

    2015-06-01

    Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying metabolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Furthermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of glycolysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher metabolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation programing, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation. PMID:25917094

  10. Stereomicroscopic 3D-pattern profiling of murine and human intestinal inflammation reveals unique structural phenotypes

    PubMed Central

    Rodriguez-Palacios, Alex; Kodani, Tomohiro; Kaydo, Lindsey; Pietropaoli, Davide; Corridoni, Daniele; Howell, Scott; Katz, Jeffry; Xin, Wei; Pizarro, Theresa T.; Cominelli, Fabio

    2015-01-01

    Histology is fundamental to assess two-dimensional intestinal inflammation; however, inflammatory bowel diseases (IBDs) are often indistinguishable microscopically on the basis of mucosal biopsies. Here, we use stereomicroscopy (SM) to rapidly profile the entire intestinal topography and assess inflammation. We examine the mucosal surface of >700 mice (encompassing >16 strains and various IBD-models), create a profiling catalogue of 3D-stereomicroscopic abnormalities and demonstrate that mice with comparable histological scores display unique sub-clusters of 3D-structure-patterns of IBD pathology, which we call 3D-stereoenterotypes, and which are otherwise indiscernible histologically. We show that two ileal IBD-stereoenterotypes (‘cobblestones' versus ‘villous mini-aggregation') cluster separately within two distinct mouse lines of spontaneous ileitis, suggesting that host genetics drive unique and divergent inflammatory 3D-structural patterns in the gut. In humans, stereomicroscopy reveals ‘liquefaction' lesions and hierarchical fistulous complexes, enriched with clostridia/segmented filamentous bacteria, running under healthy mucosa in Crohn's disease. We suggest that stereomicroscopic (3D-SMAPgut) profiling can be easily implemented and enable the comprehensive study of inflammatory 3D structures, genetics and flora in IBD. PMID:26154811

  11. Phenotypic and Genotypic Characterization of Pediococcus Strains Isolated from Human Clinical Sources

    PubMed Central

    Barros, Rosana R.; Carvalho, Maria Da Glória S.; Peralta, José Mauro; Facklam, Richard R.; Teixeira, Lúcia M.

    2001-01-01

    Seventy-two strains of pediococci isolated from human clinical sources were characterized by conventional physiological tests, chromogenic enzymatic tests, analysis of whole-cell protein profiles (WCPP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analysis of chromosomal DNA restriction profiles by pulsed-field gel electrophoresis (PFGE). Conventional tests allowed identification of 67 isolates: 52 strains were identified as Pediococcus acidilactici, 15 strains were identified as Pediococcus pentosaceus, and 5 strains were not identified because of atypical reactions. Analysis of WCPP identified all isolates since each species had a unique WCPP. By the WCPP method, the atypical strains were identified as P. acidilactici (two strains) and P. pentosaceus (three strains). The chromogenic substrate test with o-nitrophenyl-β-d-glucopyranoside differentiated all 54 strains of P. acidilactici (negative reactions) and 13 (72%) of 18 strains of P. pentosaceus (positive reactions). Isolates of both species were shown to be nonclonal as revealed by the genetic diversity when chromosomal DNA was analyzed by PFGE. Using WCPP as the definitive identification procedure, P. acidilactici (28 of 54 strains; 51.8%) was more likely than P. pentosaceus (4 of 18 strains; 22.3%) to be isolated from blood cultures. PMID:11283035

  12. Ciliated Adenosquamous Carcinoma: Expanding the Phenotypic Diversity of Human Papillomavirus-Associated Tumors.

    PubMed

    Radkay-Gonzalez, Lisa; Faquin, William; McHugh, Jonathan B; Lewis, James S; Tuluc, Madalina; Seethala, Raja R

    2016-06-01

    This study describes a unique subset of ciliated, human papillomavirus (HPV) related, adenosquamous carcinomas (AsqCA) of the head and neck that in contrast to most AsqCA, often show areas with lower grade cytonuclear features. They are comprised of largely non-keratinizing squamous cell carcinoma components with cystic change, gland formation, mucin production, and cilia in tumor cells. Seven cases of ciliated AsqCA were retrieved. Site distribution was as follows: palatine tonsil-3/7, base of tongue-1/7, and neck (unknown primary site)-3/7. Despite the occasional resemblance to mucoepidermoid carcinoma (MEC), the tumors showed focal keratinizing morphology and atypia, and all tumors were negative for MAML2 rearrangements. Oropharyngeal and neck tumors were uniformly p16 positive and showed punctate staining by in situ hybridization for high risk HPV DNA. There were two distant metastases (lung), and one tumor related death. Thus, ciliated AsqCA are HPV-associated lesions that pose unique pitfalls, closely mimicking MEC and other salivary gland tumors. These tumors add to the list of those which defy the dogma that ciliated epithelium always equates to a benign process. PMID:26411881

  13. Choline Ameliorates Disease Phenotypes in Human iPSC Models of Rett Syndrome.

    PubMed

    Chin, Eunice W M; Marcy, Guillaume; Yoon, Su-In; Ma, Dongliang; Rosales, Francisco J; Augustine, George J; Goh, Eyleen L K

    2016-09-01

    Rett syndrome (RTT) is a postnatal neurodevelopmental disorder that primarily affects girls. Mutations in the methyl-CpG-binding protein 2 (MECP2) gene account for approximately 95 % of all RTT cases. To model RTT in vitro, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of two RTT patients with different mutations (MECP2 (R306C) and MECP2 (1155Δ32)) in their MECP2 gene. We found that these iPSCs were capable of differentiating into functional neurons. Compared to control neurons, the RTT iPSC-derived cells had reduced soma size and a decreased amount of synaptic input, evident both as fewer Synapsin 1-positive puncta and a lower frequency of spontaneous excitatory postsynaptic currents. Supplementation of the culture media with choline rescued all of these defects. Choline supplementation may act through changes in the expression of choline acetyltransferase, an important enzyme in cholinergic signaling, and also through alterations in the lipid metabolite profiles of the RTT neurons. Our study elucidates the possible mechanistic pathways for the effect of choline on human RTT cell models, thereby illustrating the potential for using choline as a nutraceutical to treat RTT. PMID:27379379

  14. Certain canine weakly beta-hemolytic intestinal spirochetes are phenotypically and genotypically related to spirochetes associated with human and porcine intestinal spirochetosis.

    PubMed Central

    Duhamel, G E; Muniappa, N; Mathiesen, M R; Johnson, J L; Toth, J; Elder, R O; Doster, A R

    1995-01-01

    Four canine weakly beta-hemolytic intestinal spirochetes associated with intestinal spirochetosis (IS-associated WBHIS) were compared with IS-associated human and porcine WBHIS and the type species for Serpulina hyodysenteriae and S. innocens by using phenotypic and genotypic parameters. The IS-associated canine, human, and porcine WBHIS belonged to a phyletic group distinct from but related to previously described Serpulina type species. PMID:7559984

  15. Glut1 deficiency (G1D): Epilepsy and metabolic dysfunction in a mouse model of the most common human phenotype

    PubMed Central

    Marin-Valencia, Isaac; Good, Levi B.; Ma, Qian; Duarte, Joao; Bottiglieri, Teodoro; Sinton, Christopher M.; Heilig, Charles W.; Pascual, Juan M.

    2012-01-01

    Brain glucose supplies most of the carbon required for acetyl-coenzyme A (acetyl-CoA) generation (an important step for myelin synthesis) and for neurotransmitter production via further metabolism of acetyl-CoA in the tricarboxylic acid (TCA) cycle. However, it is not known whether reduced brain glucose transporter type I (GLUT-1) activity, the hallmark of the GLUT-1 deficiency (G1D) syndrome, leads to acetyl-CoA, TCA or neurotransmitter depletion. This question is relevant because, in its most common form in man, G1D is associated with cerebral hypomyelination (manifested as microcephaly) and epilepsy, suggestive of acetyl-CoA depletion and neurotransmitter dysfunction, respectively. Yet, brain metabolism in G1D remains underexplored both theoretically and experimentally, partly because computational models of limited brain glucose transport are subordinate to metabolic assumptions and partly because current hemizygous G1D mouse models manifest a mild phenotype not easily amenable to investigation. In contrast, adult antisense G1D mice replicate the human phenotype of spontaneous epilepsy associated with robust thalamocortical electrical oscillations. Additionally, and in consonance with human metabolic imaging observations, thalamus and cerebral cortex display the lowest GLUT-1 expression and glucose uptake in the mutant mouse. This depletion of brain glucose is associated with diminished plasma fatty acids and elevated ketone body levels, and with decreased brain acetyl-CoA and fatty acid contents, consistent with brain ketone body consumption and with stimulation of brain beta-oxidation and/or diminished cerebral lipid synthesis. In contrast with other epilepsies, astrocyte glutamine synthetase expression, cerebral TCA cycle intermediates, amino acid and amine neurotransmitter contents are also intact in G1D. The data suggest that the TCA cycle is preserved in G1D because reduced glycolysis and acetyl-CoA formation can be balanced by enhanced ketone body

  16. ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis.

    PubMed

    Arend, Peter

    2016-01-01

    The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been "lost" over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti

  17. The extent of parasite-associated necrosis in the placenta and foetal tissues of cattle following Neospora caninum infection in early and late gestation correlates with foetal death.

    PubMed

    Gibney, E Helen; Kipar, Anja; Rosbottom, Anne; Guy, Catherine S; Smith, Robert F; Hetzel, Udo; Trees, Alexander J; Williams, Diana J L

    2008-04-01

    The protozoan parasite Neospora caninum is the most frequently diagnosed abortifacient in the UK and a leading cause of abortion worldwide but the mechanisms leading to abortion are not fully understood. The distribution of parasites and the histopathological changes in the placenta and foetus were compared in 12 cows following experimental infection of cattle with N. caninum in early (n=6) and late (n=6) gestation, by PCR, immunohistology, light microscopy and transmission electron microscopy. Twelve uninfected pregnant cattle were used as controls. Infection in early gestation led to foetal death. In the placentae of cattle immediately following foetal death, N. caninum DNA was detected and there was evidence of widespread parasite dissemination. This was associated with extensive focal epithelial necrosis, serum leakage and moderate maternal interstitial mononuclear cell infiltration. In the foetuses, parasites were evident in all tissues examined and were associated with necrosis. In the placenta of cattle infected in late gestation, N. caninum DNA was detected sporadically but parasites were not evident immunohistologically. Small foci of necrosis were seen associated with mild interstitial mononuclear cell infiltration. Detection of N. caninum DNA in the foetuses was sporadic and parasites were demonstrated immunohistologically in brain and spinal cord only, with an associated mononuclear cell infiltration. This data is consistent with uncontrolled parasite spread in an immunologically immature foetus and could, via multiparenchymal necrosis of foetal tissues or the widespread necrosis and inflammation observed in the placenta, be the cause of Neospora-associated abortions. PMID:18021783

  18. Effects of Activin A on the phenotypic properties of human periodontal ligament cells.

    PubMed

    Sugii, Hideki; Maeda, Hidefumi; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Koori, Katsuaki; Hasegawa, Daigaku; Hamano, Sayuri; Yuda, Asuka; Monnouchi, Satoshi; Akamine, Akifumi

    2014-09-01

    Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily and a dimer of inhibinβa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1β expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1β or TNF-α also up-regulated the expression of the gene encoding inhibinβa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction. PMID:24928494

  19. Phenotypic Features of Circulating Leukocytes from Non-human Primates Naturally Infected with Trypanosoma cruzi Resemble the Major Immunological Findings Observed in Human Chagas Disease

    PubMed Central

    Mattoso-Barbosa, Armanda Moreira; Perdigão-de-Oliveira, Marcelo; Costa, Ronaldo Peres; Elói-Santos, Silvana Maria; Gomes, Matheus de Souza; do Amaral, Laurence Rodrigues; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Dick, Edward J.; Hubbard, Gene B.; VandeBerg, Jane F.; VandeBerg, John L.

    2016-01-01

    Background Cynomolgus macaques (Macaca fascicularis) represent a feasible model for research on Chagas disease since natural T. cruzi infection in these primates leads to clinical outcomes similar to those observed in humans. However, it is still unknown whether these clinical similarities are accompanied by equivalent immunological characteristics in the two species. We have performed a detailed immunophenotypic analysis of circulating leukocytes together with systems biology approaches from 15 cynomolgus macaques naturally infected with T. cruzi (CH) presenting the chronic phase of Chagas disease to identify biomarkers that might be useful for clinical investigations. Methods and Findings Our data established that CH displayed increased expression of CD32+ and CD56+ in monocytes and enhanced frequency of NK Granzyme A+ cells as compared to non-infected controls (NI). Moreover, higher expression of CD54 and HLA-DR by T-cells, especially within the CD8+ subset, was the hallmark of CH. A high level of expression of Granzyme A and Perforin underscored the enhanced cytotoxicity-linked pattern of CD8+ T-lymphocytes from CH. Increased frequency of B-cells with up-regulated expression of Fc-γRII was also observed in CH. Complex and imbricate biomarker networks demonstrated that CH showed a shift towards cross-talk among cells of the adaptive immune system. Systems biology analysis further established monocytes and NK-cell phenotypes and the T-cell activation status, along with the Granzyme A expression by CD8+ T-cells, as the most reliable biomarkers of potential use for clinical applications. Conclusions Altogether, these findings demonstrated that the similarities in phenotypic features of circulating leukocytes observed in cynomolgus macaques and humans infected with T. cruzi further supports the use of these monkeys in preclinical toxicology and pharmacology studies applied to development and testing of new drugs for Chagas disease. PMID:26808481

  20. Gene expression correlations in human cancer cell lines define molecular interaction networks for epithelial phenotype.

    PubMed

    Kohn, Kurt W; Zeeberg, Barry M; Reinhold, William C; Pommier, Yves

    2014-01-01

    Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute's CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions (CLDN7, CLDN4, CLDN3, MARVELD3, MARVELD2, TJP3, CGN, CRB3, LLGL2, EPCAM, LNX1); interactions at adherens junctions (CDH1, ADAP1, CAMSAP3); interactions at desmosomes (PPL, PKP3, JUP); transcription regulation of cell-cell junction complexes (GRHL1 and 2); epithelial RNA splicing regulators (ESRP1 and 2); epithelial vesicle traffic (RAB25, EPN3, GRHL2, EHF, ADAP1, MYO5B); epithelial Ca(+2) signaling (ATP2C2, S100A14, BSPRY); terminal differentiation of epithelial cells (OVOL1 and 2, ST14, PRSS8, SPINT1 and 2); maintenance of apico-basal polarity (RAB25, LLGL2, EPN3). The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets. PMID:24940735

  1. Expression of CD39 by human peripheral blood CD4+CD25+ T cells denotes a regulatory memory phenotype

    PubMed Central

    Dwyer, Karen M.; Hanidziar, Dusan; Putheti, Prabhakar; Hill, Prue A; Pommey, Sandra; McRae, Jennifer L; Winterhalter, Adam; Doherty, Glen; Deaglio, Silvia; Koulmanda, Maria; Gao, Wenda; Robson, Simon C.; Strom, Terry B.

    2010-01-01

    We have shown that CD39 and CD73 are co-expressed on the surface of murine CD4+Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood derived human CD4+CD25+CD127lo T regulatory cells (Treg), defined by robust expression of Foxp3. A further distinct population of CD4+CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete pro-inflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+CD25+CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell populations to allow tracking of these in health and disease, as in renal allograft rejection. PMID:20977632

  2. Stimulators of Mineralization Limit the Invasive Phenotype of Human Osteosarcoma Cells by a Mechanism Involving Impaired Invadopodia Formation

    PubMed Central

    Cmoch, Anna; Podszywalow-Bartnicka, Paulina; Palczewska, Malgorzata; Piwocka, Katarzyna; Groves, Patrick; Pikula, Slawomir

    2014-01-01

    Background Osteosarcoma (OS) is a highly aggressive bone cancer affecting children and young adults. Growing evidence connects the invasive potential of OS cells with their ability to form invadopodia (structures specialized in extracellular matrix proteolysis). Results In this study, we tested the hypothesis that commonly used in vitro stimulators of mineralization limit the invadopodia formation in OS cells. Here we examined the invasive potential of human osteoblast-like cells (Saos-2) and osteolytic-like (143B) OS cells treated with the stimulators of mineralization (ascorbic acid and B-glycerophosphate) and observed a significant difference in response of the tested cells to the treatment. In contrast to 143B cells, osteoblast-like cells developed a mineralization phenotype that was accompanied by a decreased proliferation rate, prolongation of the cell cycle progression and apoptosis. On the other hand, stimulators of mineralization limited osteolytic-like OS cell invasiveness into collagen matrix. We are the first to evidence the ability of 143B cells to degrade extracellular matrix to be driven by invadopodia. Herein, we show that this ability of osteolytic-like cells in vitro is limited by stimulators of mineralization. Conclusions Our study demonstrates that mineralization competency determines the invasive potential of cancer cells. A better understanding of the molecular mechanisms by which stimulators of mineralization regulate and execute invadopodia formation would reveal novel clinical targets for treating osteosarcoma. PMID:25314307

  3. Granulocytic Myeloid-Derived Suppressor Cells Accumulate in Human Placenta and Polarize toward a Th2 Phenotype.

    PubMed

    Köstlin, Natascha; Hofstädter, Kathrin; Ostermeir, Anna-Lena; Spring, Bärbel; Leiber, Anja; Haen, Susanne; Abele, Harald; Bauer, Peter; Pollheimer, Jürgen; Hartl, Dominik; Poets, Christian F; Gille, Christian

    2016-02-01

    Tolerance induction toward the semiallogeneic fetus is crucial to enable a successful pregnancy; its failure is associated with abortion or preterm delivery. Skewing T cell differentiation toward a Th2-dominated phenotype seems to be pivotal in maternal immune adaption, yet underlying mechanisms are incompletely understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that mediate T cell suppression and are increased in cord blood of healthy newborns and in peripheral blood of pregnant women. In this study, we demonstrate that granulocytic MDSCs (GR-MDSCs) accumulate in human placenta of healthy pregnancies but are diminished in patients with spontaneous abortions. Placental GR-MDSCs effectively suppressed T cell responses by expression of arginase I and production of reactive oxygen species and were activated at the maternal-fetal interface through interaction with trophoblast cells. Furthermore, GR-MDSCs isolated from placenta polarized CD4(+) T cells toward a Th2 cytokine response. These results highlight a potential role of GR-MDSCs in inducing and maintaining maternal-fetal tolerance and suggest them as a promising target for therapeutic manipulation of pregnancy complications. PMID:26712947

  4. Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches.

    PubMed

    Nizzardo, Monica; Simone, Chiara; Dametti, Sara; Salani, Sabrina; Ulzi, Gianna; Pagliarani, Serena; Rizzo, Federica; Frattini, Emanuele; Pagani, Franco; Bresolin, Nereo; Comi, Giacomo; Corti, Stefania

    2015-01-01

    Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders. PMID:26123042

  5. Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer's Disease Phenotypes.

    PubMed

    Raja, Waseem K; Mungenast, Alison E; Lin, Yuan-Ta; Ko, Tak; Abdurrob, Fatema; Seo, Jinsoo; Tsai, Li-Huei

    2016-01-01

    The dismal success rate of clinical trials for Alzheimer's disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD. PMID:27622770

  6. Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells.

    PubMed

    Schildknecht, Stefan; Pöltl, Dominik; Nagel, Daniel M; Matt, Florian; Scholz, Diana; Lotharius, Julie; Schmieg, Nathalie; Salvo-Vargas, Alberto; Leist, Marcel

    2009-11-15

    LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP(+)) toxicity at < or =5 microM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP(+) toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-l-tyrosine or 3-iodo-l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP(+)-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP(+)-treated LUHMES. PMID:19647008

  7. Genomic Features of the Human Dopamine Transporter Gene and Its Potential Epigenetic States: Implications for Phenotypic Diversity

    SciTech Connect

    Shumay, E.; Shumay, E.; Fowler, J.S.; Volkow, N.D.

    2010-06-01

    Human dopamine transporter gene (DAT1 or SLC6A3) has been associated with various brain-related diseases and behavioral traits and, as such, has been investigated intensely in experimental- and clinical-settings. However, the abundance of research data has not clarified the biological mechanism of DAT regulation; similarly, studies of DAT genotype-phenotype associations yielded inconsistent results. Hence, our understanding of the control of the DAT protein product is incomplete; having this knowledge is critical, since DAT plays the major role in the brain's dopaminergic circuitry. Accordingly, we reevaluated the genomic attributes of the SLC6A3 gene that might confer sensitivity to regulation, hypothesizing that its unique genomic characteristics might facilitate highly dynamic, region-specific DAT expression, so enabling multiple regulatory modes. Our comprehensive bioinformatic analyzes revealed very distinctive genomic characteristics of the SLC6A3, including high inter-individual variability of its sequence (897 SNPs, about 90 repeats and several CNVs spell out all abbreviations in abstract) and pronounced sensitivity to regulation by epigenetic mechanisms, as evident from the GC-bias composition (0.55) of the SLC6A3, and numerous intragenic CpG islands (27 CGIs). We propose that this unique combination of the genomic features and the regulatory attributes enables the differential expression of the DAT1 gene and fulfills seemingly contradictory demands to its regulation; that is, robustness of region-specific expression and functional dynamics.

  8. Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells

    SciTech Connect

    Schildknecht, Stefan; Poeltl, Dominik; Nagel, Daniel M.; Matt, Florian; Scholz, Diana; Lotharius, Julie; Schmieg, Nathalie; Salvo-Vargas, Alberto; Leist, Marcel

    2009-11-15

    LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP{sup +}) toxicity at <= 5 muM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP{sup +} toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-L-tyrosine or 3-iodo-L-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP{sup +}-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP{sup +}-treated LUHMES.

  9. Concealed expansion of immature precursors underpins acute burst of adult HSC activity in foetal liver.

    PubMed

    Rybtsov, Stanislav; Ivanovs, Andrejs; Zhao, Suling; Medvinsky, Alexander

    2016-04-15

    One day prior to mass emergence of haematopoietic stem cells (HSCs) in the foetal liver at E12.5, the embryo contains only a few definitive HSCs. It is thought that the burst of HSC activity in the foetal liver is underpinned by rapid maturation of immature embryonic precursors of definitive HSCs, termed pre-HSCs. However, because pre-HSCs are not detectable by direct transplantations into adult irradiated recipients, the size and growth of this population, which represents the embryonic rudiment of the adult haematopoietic system, remains uncertain. Using a novel quantitative assay, we demonstrate that from E9.5 the pre-HSC pool undergoes dramatic growth in the aorta-gonad-mesonephros region and by E11.5 reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Thus, this study provides for the first time a quantitative basis for our understanding of how the large population of definitive HSCs emerges in the foetal liver. PMID:27095492

  10. Secondary School Experiences of Individuals with Foetal Alcohol Spectrum Disorder: Perspectives of Parents and Their Children

    ERIC Educational Resources Information Center

    Duquette, Cheryll; Stodel, Emma; Fullarton, Stephanie; Hagglund, Karras

    2007-01-01

    The purpose of this study was to examine persistence in school among students with Foetal Alcohol Spectrum Disorder (FASD) from the perspectives of the students themselves and their parents. Tinto's (1975, 1997) Student Integration Model (SIM) provided the theoretical framework for this research. This model involves an interplay between (1)…

  11. Caregiver Perceptions of the Community Integration of Adults with Foetal Alcohol Spectrum Disorder in British Columbia

    ERIC Educational Resources Information Center

    Clark, Erica; Minnes, Patricia; Lutke, Jan; Ouellette-Kuntz, Helene

    2008-01-01

    Background: Adults with foetal alcohol spectrum disorder (FASD) require support to be part of the community; however, most have few supports other than family and friends. The purpose of this study was to assess caregiver perceptions of community integration of adults with FASD living in British Columbia. Method: The Assimilation, Integration,…

  12. Pedagogically Bereft! Improving Learning Outcomes for Children with Foetal Alcohol Spectrum Disorders

    ERIC Educational Resources Information Center

    Carpenter, Barry

    2011-01-01

    Foetal alcohol spectrum disorder (FASD) is the most common non-genetic cause of learning disability, affecting around 1% of live births in Europe, and costing an estimated $2.9 million per individual across their lifespan. In adulthood, non-reversible brain damage is often compounded by secondary disabilities in adulthood, such as mental health…

  13. Foetal magnetocardiography with a multi-channel HTS rf SQUID gradiometer

    NASA Astrophysics Data System (ADS)

    Zhang, Y.; Wolters, N.; Lomparski, D.; Zander, W.; Banzet, M.; Schubert, J.; Krause, H.-J.; Geue, D.; van Leeuwen, P.

    2006-05-01

    Foetal magnetocardiography (fMCG) is a promising technique for prenatal cardiac diagnosis. In this paper, we discuss the special requirements of a system for fMCG measurements. A SQUID system incorporating five HTS rf SQUID magnetometers has been developed and constructed. Four magnetometers are arranged at the bottom of the cryostat in a 4 cm × 4 cm square configuration. One reference magnetometer is located 20 cm above the plane to form four first-order axial gradiometers with the four bottom magnetometers. The magnetometer with a 18 mm diameter flux focuser, which reaches a field sensitivity of 20-30 fT Hz-1/2, proved sufficient for recording foetal magnetocardiograms in a magnetically shielded room. The cardiac activities of two foetuses (31st and 33rd weeks of gestation) were recorded in 90 Hz bandwidth. The foetal QRS peak signals ranged from 4 to 7 pT and could easily be identified in the real-time gradiometer outputs with a signal-to-noise ratio of up to five. Furthermore, the averaged fMCG data enabled the determination of de- and repolarization time intervals. Overall, the fMCG signals proved of sufficient quality to perform foetal heart diagnostics.

  14. Concealed expansion of immature precursors underpins acute burst of adult HSC activity in foetal liver

    PubMed Central

    Ivanovs, Andrejs; Zhao, Suling; Medvinsky, Alexander

    2016-01-01

    One day prior to mass emergence of haematopoietic stem cells (HSCs) in the foetal liver at E12.5, the embryo contains only a few definitive HSCs. It is thought that the burst of HSC activity in the foetal liver is underpinned by rapid maturation of immature embryonic precursors of definitive HSCs, termed pre-HSCs. However, because pre-HSCs are not detectable by direct transplantations into adult irradiated recipients, the size and growth of this population, which represents the embryonic rudiment of the adult haematopoietic system, remains uncertain. Using a novel quantitative assay, we demonstrate that from E9.5 the pre-HSC pool undergoes dramatic growth in the aorta-gonad-mesonephros region and by E11.5 reaches the size that matches the number of definitive HSCs in the E12.5 foetal liver. Thus, this study provides for the first time a quantitative basis for our understanding of how the large population of definitive HSCs emerges in the foetal liver. PMID:27095492

  15. "One More for My Baby": Foetal Alcohol Syndrome and Its Implications for Social Workers

    ERIC Educational Resources Information Center

    Cousins, Wendy; Wells, Karen

    2005-01-01

    Foetal alcohol syndrome has been described as the commonest preventable cause of mental retardation in the Western world. It refers to a pattern of malformations, growth retardation and central nervous system impairments found in children of mothers who drink large amounts of alcohol while they are pregnant. This paper describes the nature of…

  16. Foetal scalp blood sampling during labour for pH and lactate measurements.

    PubMed

    Carbonne, Bruno; Pons, Kelly; Maisonneuve, Emeline

    2016-01-01

    Second-line methods of foetal monitoring have been developed in an attempt to reduce unnecessary interventions due to continuous cardiotocography (CTG), and to better identify foetuses that are at risk of intrapartum asphyxia. Very few studies directly compared CTG with foetal scalp blood (FBS) and CTG only. Only one randomised controlled trial (RCT) was published in the 1970s and had limited power to assess neonatal outcome. Direct and indirect comparisons conclude that FBS could reduce the number of caesarean deliveries associated with the use of continuous CTG. The main drawbacks of FBS are its invasive and discontinuous nature and the need for a sufficient volume of foetal blood for analysis, especially for pH measurement, resulting in failure rates reaching 10%. FBS for lactate measurement became popular with the design of test-strip devices, requiring <0.5 mL of foetal blood. RCTs showed similar outcomes with the use of FBS for lactates compared with pH in terms of obstetrical interventions and neonatal outcomes. In conclusion, there is some evidence that FBS reduces the need for operative deliveries. However, the evidence is limited with regard to actual standards, and large RCTs, directly comparing CTG only with CTG with FBS, are still needed. PMID:26253238

  17. Frequency and Time Domain Analysis of Foetal Heart Rate Variability with Traditional Indexes: A Critical Survey

    PubMed Central

    Romano, Maria; Iuppariello, Luigi; Ponsiglione, Alfonso Maria; Improta, Giovanni; Bifulco, Paolo; Cesarelli, Mario

    2016-01-01

    Monitoring of foetal heart rate and its variability (FHRV) covers an important role in assessing health of foetus. Many analysis methods have been used to get quantitative measures of FHRV. FHRV has been studied in time and in frequency domain and interesting clinical results have been obtained. Nevertheless, a standardized definition of FHRV and a precise methodology to be used for its evaluation are lacking. We carried out a literature overview about both frequency domain analysis (FDA) and time domain analysis (TDA). Then, by using simulated FHR signals, we defined the methodology for FDA. Further, employing more than 400 real FHR signals, we analysed some of the most common indexes, Short Term Variability for TDA and power content of the spectrum bands and sympathovagal balance for FDA, and evaluated their ranges of values, which in many cases are a novelty. Finally, we verified the relationship between these indexes and two important parameters: week of gestation, indicator of foetal growth, and foetal state, classified as active or at rest. Our results indicate that, according to literature, it is necessary to standardize the procedure for FHRV evaluation and to consider week of gestation and foetal state before FHR analysis. PMID:27195018

  18. Frequency and Time Domain Analysis of Foetal Heart Rate Variability with Traditional Indexes: A Critical Survey.

    PubMed

    Romano, Maria; Iuppariello, Luigi; Ponsiglione, Alfonso Maria; Improta, Giovanni; Bifulco, Paolo; Cesarelli, Mario

    2016-01-01

    Monitoring of foetal heart rate and its variability (FHRV) covers an important role in assessing health of foetus. Many analysis methods have been used to get quantitative measures of FHRV. FHRV has been studied in time and in frequency domain and interesting clinical results have been obtained. Nevertheless, a standardized definition of FHRV and a precise methodology to be used for its evaluation are lacking. We carried out a literature overview about both frequency domain analysis (FDA) and time domain analysis (TDA). Then, by using simulated FHR signals, we defined the methodology for FDA. Further, employing more than 400 real FHR signals, we analysed some of the most common indexes, Short Term Variability for TDA and power content of the spectrum bands and sympathovagal balance for FDA, and evaluated their ranges of values, which in many cases are a novelty. Finally, we verified the relationship between these indexes and two important parameters: week of gestation, indicator of foetal growth, and foetal state, classified as active or at rest. Our results indicate that, according to literature, it is necessary to standardize the procedure for FHRV evaluation and to consider week of gestation and foetal state before FHR analysis. PMID:27195018

  19. Induction of lethal bystander effects in human breast cancer cell cultures by DNA-incorporated Iodine-125 depends on phenotype

    PubMed Central

    Akudugu, John M.; Azzam, Edouard I.; Howell, Roger W.

    2013-01-01

    Purpose This study uses a three-dimensional cell culture model to investigate lethal bystander effects in human breast cancer cell cultures (MCF-7, MDA-MB-231) treated with 125I-labeled 5-iodo-2′-deoxyuridine (125IdU). These breast cancer cell lines respectively form metastatic xenografts in nude mice in an estrogen-dependent and independent manner. Materials and methods In the present study, these cells were cultured in loosely-packed three-dimensional architecture in a Cytomatrix™ carbon scaffold. Cultures were pulse-labeled for 3 h with 125IdU to selectively irradiate a minor fraction of cells, and simultaneously co-pulse-labeled with 0.04 mM 5-ethynyl-2′-deoxyuridine (EdU) to identify the radiolabeled cells using Click-iT® EdU and flow cytometry. The cultures were then washed and incubated for 48 h. The cells were then harvested, serially diluted, and seeded for colony formation. Aliquots of cells were subjected to flow cytometry to determine the percentage of cells labeled with 125IdU/EdU. Additional aliquots were used to determine the mean 125I activity per labeled cell. The percentage of labeled cells was about 15% and 10% for MCF-7 and MDA-MB-231 cells, respectively. This created irradiation conditions wherein the cross-dose to unlabeled cells was small relative to the self-dose to labeled cells. The surviving fraction relative to EdU-treated controls was measured. Results Survival curves indicated significant lethal bystander effect in MCF-7 cells, however, no significant lethal bystander effect was observed in MDA-MB-231 cells. Conclusions These studies demonstrate the capacity of 125IdU to induce lethal bystander effects in human breast cancer cells and suggest that the response depends on phenotype. PMID:22489958

  20. A Comprehensive Overview of Skeletal Phenotypes Associated with Alterations in Wnt/β-catenin Signaling in Humans and Mice

    PubMed Central

    Maupin, Kevin A.; Droscha, Casey J.; Williams, Bart O.

    2013-01-01

    The Wnt signaling pathway plays key roles in differentiation and development and alterations in this signaling pathway are causally associated with numerous human diseases. While several laboratories were examining roles for Wnt signaling in skeletal development during the 1990s, interest in the pathway rose exponentially when three key papers were published in 2001–2002. One report found that loss of the Wnt co-receptor, Low-density lipoprotein related protein-5 (LRP5), was the underlying genetic cause of the syndrome Osteoporosis pseudoglioma (OPPG). OPPG is characterized by early-onset osteoporosis causing increased susceptibility to debilitating fractures. Shortly thereafter, two groups reported that individuals carrying a specific point mutation in LRP5 (G171V) develop high-bone mass. Subsequent to this, the causative mechanisms for these observations heightened the need to understand the mechanisms by which Wnt signaling controlled bone development and homeostasis and encouraged significant investment from biotechnology and pharmaceutical companies to develop methods to activate Wnt signaling to increase bone mass to treat osteoporosis and other bone disease. In this review, we will briefly summarize the cellular mechanisms underlying Wnt signaling and discuss the observations related to OPPG and the high-bone mass disorders that heightened the appreciation of the role of Wnt signaling in normal bone development and homeostasis. We will then present a comprehensive overview of the core components of the pathway with an emphasis on the phenotypes associated with mice carrying genetically engineered mutations in these genes and clinical observations that further link alterations in the pathway to changes in human bone. PMID:26273492

  1. Effects of the differentiated keratinocyte phenotype on expression levels of CYP1-4 family genes in human skin cells

    SciTech Connect

    Du Liping; Neis, Mark M.; Ladd, Patricia A.; Yost, Garold S.; Keeney, Diane S. . E-mail: diane.keeney@vanderbilt.edu

    2006-06-01

    Epoxyeicosatrienoic acids produced by mouse CYP2B19 have been implicated in mechanisms regulating epidermal cornification (Ladd, P.A., Du, L., Capdevila, J.H., Mernaugh, R., Keeney, D.S., 2003. Epoxyeicosatrienoic acids activate transglutaminases in situ and induce cornification of epidermal keratinocytes. J. Biol. Chem. 278, 35184-35192). In this study, we aimed to identify CYPs that are up-regulated during keratinocyte differentiation and potentially responsible for epoxyeicosatrienoic acid formation in human skin. The cellular differentiation state of human epidermal cell cultures was manipulated to resemble the basal, spinous, and granular cell phenotypes in vivo. Changes in CYP mRNA levels were measured as a function of differentiation state for a panel of 15 CYPs that included known and putative arachidonate monooxygenases. Quantitative real-time PCR analyses showed that all of the CYPs were expressed in differentiating epidermal cell cultures and in human epidermis, with the exception of CYP2B6, which was poorly expressed in vitro. Six CYPs were strongly up-regulated at Day 6 and Day 8 of in vitro differentiation (CYP4B1, 2W1, 2C18, 3A4, 2C19, 2C9); the increase in mRNA levels ranged from 27- to 356-fold. Only CYP2U1 mRNA levels decreased (6-fold change) during cellular differentiation. Six CYPs showed little variation (<2-fold change) in mRNA levels during in vitro differentiation (CYP2S1, 2J2, 1B1, 1A1, 2E1, 2D6). No single CYP was identifiable as being a functional counterpart to CYP2B19 in mouse skin since none qualified as being mainly responsible for epidermal epoxyeicosatrienoic acid formation. Rather, the data suggest that epoxyeicosatrienoic acids in human skin are formed by several CYPs expressed in different cell layers of the epidermis. This would predict that CYP-derived eicosanoids have different functions in different epidermal cell layers.

  2. Mutations of the KIT (Mast/Stem cell growth factor receptor) proto-oncogene account for a continuous range of phenotypes in human piebaldism

    SciTech Connect

    Spritz, R.A.; Holmes, S.A. ); Ramesar, R.; Greenberg, J.; Beighton, P.; Curtis, D.

    1992-11-01

    Piebaldism is a rare autosomal dominant disorder of pigmentation, characterized by congenital patches of white skin and hair from which melanocytes are absent. The authors have previously shown that piebaldism can result from missense and frameshift mutations of the KIT proto-oncogene, which encodes the cellular receptor tyrosine kinase for the mast/stem cell growth factor. Here, the authors report two novel KIT mutations associated with human piebaldism. A proximal frameshift is associated with a mild piebald phenotype, and a splice-junction mutation is associated with a highly variable piebald phenotype. They discuss the apparent relationship between the predicted impact of specific KIT mutations on total KIT-dependent signal transduction and the severity of the resultant piebald phenotypes. 35 refs., 5 figs.

  3. Human Cardiac Mesenchymal Stromal Cells with CD105+CD34- Phenotype Enhance the Function of Post-Infarction Heart in Mice

    PubMed Central

    Wiśniewska, Ewa; Jarosz-Biej, Magdalena; Smolarczyk, Ryszard; Cichoń, Tomasz; Głowala-Kosińska, Magdalena; Śliwka, Joanna; Garbacz, Marcin; Szczypior, Mateusz; Jaźwiec, Tomasz; Langrzyk, Agnieszka; Zembala, Michał; Szala, Stanisław

    2016-01-01

    Aims The aim of the present study was to isolate mesenchymal stromal cells (MSC) with CD105+CD34- phenotype from human hearts, and to investigate their therapeutic potential in a mouse model of hindlimb ischemia and myocardial infarction (MI). The study aimed also to investigate the feasibility of xenogeneic MSCs implantation. Methods and Results MSC isolated from human hearts were multipotent cells. Separation of MSC with CD105+CD34- phenotype limited the heterogeneity of the originally isolated cell population. MSC secreted a number of anti-inflammatory and proangiogenic cytokines (mainly IL-6, IL-8, and GRO). Human MSC were transplanted into C57Bl/6NCrl mice. Using the mouse model of hindlimb ischemia it was shown that human MSC treated mice demonstrated a higher capillary density 14 days after injury. It was also presented that MSC administrated into the ischemic muscle facilitated fast wound healing (functional recovery by ischemic limb). MSC transplanted into an infarcted myocardium reduced the post-infarction scar, fibrosis, and increased the number of blood vessels both in the border area, and within the post-infarction scar. The improvement of left ventricular ejection fraction was also observed. Conclusion In two murine models (hindlimb ischemia and MI) we did not observe the xenotransplant rejection. Indeed, we have shown that human cardiac mesenchymal stromal cells with CD105+CD34- phenotype exhibit therapeutic potential. It seems that M2 macrophages are essential for healing and repair of the post-infarcted heart. PMID:27415778

  4. Association between Maternal Pelvis Height and Intrapartum Foetal Head Moulding in Ugandan Mothers with Spontaneous Vertex Deliveries

    PubMed Central

    Luboga, Samuel Abilemech; Luboobi, Livingstone; Mirembe, Florence

    2016-01-01

    Introduction. In Sub-Saharan Africa, excessive foetal head moulding is commonly associated with cephalopelvic disproportion and obstructed labour. This study set out to determine the associations of maternal pelvis height and maternal height with intrapartum foetal head moulding. Methods. This was a multisite secondary analysis of maternal birth records of mothers with singleton pregnancies ending in a spontaneous vertex delivery. A summary of the details of the pregnancy and delivery records were reviewed and analysed using multilevel logistic regression respect to foetal head moulding. The alpha level was set at P < 0.05. Results. 412 records were obtained, of which 108/385 (28%) observed foetal head moulding. There was a significant reduction in risk of foetal head moulding with increasing maternal height (Adj. IRR 0.97, P = 0.05), maternal pelvis height (Adj. IRR 0.88, P < 0.01), and raptured membranes (Adj. IRR 0.10, P < 0.01). There was a significant increased risk of foetal head moulding with increasing birth weight (Adj. IRR 1.90, P < 0.01) and duration of monitored active labour (Adj. IRR 1.21, P < 0.01) in the final model. Conclusion. This study showed that increasing maternal height and maternal pelvis height were associated with a significant reduction in intrapartum foetal head moulding. PMID:27034678

  5. T-cell function, T-cell phenotype and its role in responsiveness to recombinant human erythropoietin in hemodialysis patients.

    PubMed

    Abdalla, M; Yassein, S; Darwish, K H

    2010-01-01

    Resistance to recombinant human erythropoietin (Epo) occurs in a small proportion of hemodialysis (HD) patients. In this study we investigated the relationship between T-cell phenotype (using flow cytometry), T-cell function (by measuring in vitro cytokine production) and responsiveness to Epo in HD patients and to compare the results with those from healthy controls. T-cell phenotypes were assessed and T-cell function was studied. The study included 24 chronic renal failure (CRF) patients on HD treated with rHuEPO as well as 14 normal control subjects. Dual-colour immunofluorescence and flow cytometry were used to compare the surface antigen expression on freshly isolated CD4+ and CD8+ T-cells from PBMC of the studied groups. Levels of a panel of selected cytokines (IL-4, IFN-gamma, slL-2R and IL-10) were determined in PBMC culture supernatants and in plasma samples (TNF-alpha, IFN-gamma, IL-6, slL-2R) using (ELISA) kits. Patients were followed-up for 24 months and a survival study was carried out. T-cells from poor responders showed increased proportions of CD4+/CD28- cells and CD8+/CD28- cells compared with both good responders and controls. Compared with their CD28+ counterparts, CD4+/CD28- T-cells produced significantly more IFN-gamma, enabling them to function as pro-inflammatory cells. There was no difference in secretion of IFN-gamma, slL-2R or IL-4 in PBMC cultures obtained from HD patients and controls. However, Unstimulated PBMC from poor responders generated increased levels of IL-10 poor compared with both good responders and controls. Plasma slL-2R and IL-6 were significantly elevated in both good and poor responders compared with controls. Plasma levels of IFN-gamma and TNF-alpha were undetectable in both HD patients and controls. In the follow up period, more deaths were occurring among the poor responders than the good responders. Based on the finding of the this study we may suggest that, in the absence of any obvious cause, poor response to Epo

  6. Foetal antiepileptic drug exposure and verbal versus non-verbal abilities at three years of age

    PubMed Central

    Meador, Kimford J.; Baker, Gus A.; Browning, Nancy; Cohen, Morris J.; Clayton-Smith, Jill; Kalayjian, Laura A.; Kanner, Andres; Liporace, Joyce D.; Pennell, Page B.; Privitera, Michael

    2011-01-01

    We previously reported that foetal valproate exposure impairs intelligence quotient. In this follow-up investigation, we examined dose-related effects of foetal antiepileptic drug exposure on verbal and non-verbal cognitive measures. This investigation is an ongoing prospective observational multi-centre study in the USA and UK, which has enrolled pregnant females with epilepsy on monotherapy from 1999 to 2004. The study seeks to determine if differential long-term neurodevelopmental effects exist across four commonly used drugs (carbamazepine, lamotrigine, phenytoin and valproate). This report compares verbal versus non-verbal cognitive outcomes in 216 children who completed testing at the age of three years. Verbal and non-verbal index scores were calculated from the Differential Ability Scales, Preschool Language Scale, Peabody Picture Vocabulary Test and Developmental Test of Visual-Motor Integration. Verbal abilities were lower than non-verbal in children exposed in utero to each drug. Preconceptional folate use was associated with higher verbal outcomes. Valproate was associated with poorer cognitive outcomes. Performance was negatively associated with valproate dose for both verbal and non-verbal domains and negatively associated with carbamazepine dose for verbal performance. No dose effects were seen for lamotrigine and phenytoin. Since foetal antiepileptic drug exposure is associated with lower verbal than non-verbal abilities, language may be particularly susceptible to foetal exposure. We hypothesize that foetal drug exposure may alter normal cerebral lateralization. Further, a dose-dependent relationship is present for both lower verbal and non-verbal abilities with valproate and for lower verbal abilities with carbamazepine. Preconceptional folate may improve cognitive outcomes. Additional research is needed to confirm these findings, extend the study to other drugs, define the risks associated with drug treatment for seizures in the neonates, and

  7. MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

    SciTech Connect

    Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

    2013-05-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC

  8. Preserved functional autonomic phenotype in adult mice overexpressing moderate levels of human alpha‐synuclein in oligodendrocytes

    PubMed Central

    Tank, Jens; da Costa‐Goncalves, Andrey C.; Kamer, Ilona; Qadri, Fatimunnisa; Ubhi, Kiren; Rockenstein, Edward; Diedrich, André; Masliah, Eliezer; Gross, Volkmar; Jordan, Jens

    2014-01-01

    Abstract Mice overexpressing human alpha‐synuclein in oligodendrocytes (MBP1‐α‐syn) recapitulate some key functional and neuropathological features of multiple system atrophy (MSA). Whether or not these mice develop severe autonomic failure, which is a key feature of human MSA, remains unknown. We explored cardiovascular autonomic regulation using long‐term blood pressure (BP) radiotelemetry and pharmacological testing. We instrumented 12 MBP1‐α‐syn mice and 11 wild‐type mice aged 9 months for radiotelemetry. Animals were tested with atropine, metoprolol, clonidine, and trimethaphan at 9 and 12 months age. We applied spectral and cross‐spectral analysis to assess heart rate (HR) and BP variability. At 9 months of age daytime BP (transgenic: 101 ± 2 vs. wild type: 99 ± 2 mmHg) and HR (497 ± 11 vs. 505 ± 16 beats/min) were similar. Circadian BP and HR rhythms were maintained. Nighttime BP (109 ± 2 vs. 108 ± 2 mmHg) and HR (575 ± 15 vs. 569 ± 14 beats/min), mean arterial BP responses to trimethaphan (−21 ± 8 vs. −10 ± 5 mmHg, P = 0.240) and to clonidine (−8 ± 3 vs. −5 ± 2 mmHg, P = 0.314) were similar. HR responses to atropine (+159 ± 24 vs. +146 ± 22 beats/min), and to clonidine (−188 ± 21 vs. −163 ± 33 beats/min) did not differ between strains. Baroreflex sensitivity (4 ± 1 vs. 4 ± 1 msec/mmHg) and HR variability (total power, 84 ± 17 vs. 65 ± 21 msec²) were similar under resting conditions and during pharmacological testing. Repeated measurements at 12 months of age provided similar results. In mice, moderate overexpression of human alpha‐synuclein in oligodendrocytes is not sufficient to induce overt autonomic failure. Additional mechanisms may be required to express the autonomic failure phenotype including higher levels of expression or more advanced age. PMID:25428949

  9. Pharmacological screening of bryophyte extracts that inhibit growth and induce abnormal phenotypes in human HeLa cancer cells.

    PubMed

    Krzaczkowski, Lucie; Wright, Michel; Rebérioux, Delphine; Massiot, Georges; Etiévant, Chantal; Gairin, Jean Edouard

    2009-08-01

    Antitumor activities of substances from natural sources apart from vascular plants and micro-organisms have been poorly investigated. Here we report on a pharmacological screening of a bryophyte extract library using a phenotypic cell-based assay revealing microtubules, centrosomes and DNA. Among the 219 moss extracts tested, we identified 41 extracts acting on cell division with various combinations of significant effects on interphasic and mitotic cells. Seven extracts were further studied using a cell viability assay, cell cycle analysis and the phenotypic assay. Three distinct pharmacological patterns were identified including two unusual phenotypes. PMID:19709324

  10. Epithelial Ovarian Cancer-Induced Angiogenic Phenotype of Human Omental Microvascular Endothelial Cells May Occur Independently of VEGF Signaling12

    PubMed Central

    Winiarski, Boleslaw K; Wolanska, Katarzyna I; Rai, Srijana; Ahmed, Tahanver; Acheson, Nigel; Gutowski, Nicholas J; Whatmore, Jacqueline L

    2013-01-01

    Epithelial ovarian cancer (EOC) metastasizes transcoelomically to the peritoneum and omentum, and despite surgery and chemotherapy, recurrent disease is likely. Metastasis requires the induction of proangiogenic changes in the omental microenvironment and EOC-induced omental angiogenesis is currently a key therapeutic target. In particular, antiangiogenic therapies targeting the vascular endothelial growth factor A (VEGFA) pathway are commonly used, although, with limited effects. Here, using human omental microvascular endothelial cells (HOMECs) and ovarian cancer cell lines as an in vitro model, we show that factors secreted from EOC cells increased proliferation, migration, and tube-like structure formation in HOMECs. However, EOC-induced angiogenic tube-like formation and migration were unaffected by inhibition of tyrosine kinase activity of VEGF receptors 1 and 2 (Semaxanib; SU5416) or neutralization of VEGFA (neutralizing anti-VEGFA antibody), although VEGFA165-induced HOMEC migration and tube-like structure formation were abolished. Proteomic investigation of the EOC secretome identified several alternative angiogenesis-related proteins. We screened these for their ability to induce an angiogenic phenotype in HOMECs, i.e., proliferation, migration, and tube-like structure formation. Hepatocyte growth factor (HGF) and insulin-like growth factor binding protein 7 (IGFBP-7) increased all three parameters, and cathepsin L (CL) increased migration and tubule formation. Further investigation confirmed expression of the HGF receptor c-Met in HOMECs. HGF- and EOC-induced proliferation and angiogenic tube structure formation were blocked by the c-Met inhibitor PF04217903. Our results highlight key alternative angiogenic mediators for metastatic EOC, namely, HGF, CL, and IGFBP-7, suggesting that effective antiangiogenic therapeutic strategies for this disease require inhibition of multiple angiogenic pathways. PMID:24466373

  11. Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases

    PubMed Central

    Ritzerfeld, Julia; Remmele, Steffen; Wang, Tao; Temmerman, Koen; Brügger, Britta; Wegehingel, Sabine; Tournaviti, Stella; Strating, Jeroen R.P.M.; Wieland, Felix T.; Neumann, Beate; Ellenberg, Jan; Lawerenz, Chris; Hesser, Jürgen; Erfle, Holger; Pepperkok, Rainer; Nickel, Walter

    2011-01-01

    SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes. PMID:21795383

  12. Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO2-Requiring Phenotype 1

    PubMed Central

    Price, G. D.; Badger, M. R.

    1989-01-01

    Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (Ci) and low Ci grown cells leads the creation of a high Ci requiring phenotype causing: (a) a dramatic increase in the K0.5 (Ci) for photosynthesis, (b) a loss of the ability to accumulate internal Ci, and (c) a decrease in the lag between the initial Ci accumulation following illumination and the efflux of CO2 from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO2 concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and Ci accumulation is developed in which the carboxysome plays a central role as both the site of CO2 generation from HCO3− and a resistance barrier to CO2 efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells. Images Figure 7 PMID:16667062

  13. G-CSF regulates macrophage phenotype and associates with poor overall survival in human triple-negative breast cancer

    PubMed Central

    Hollmén, Maija; Karaman, Sinem; Schwager, Simon; Lisibach, Angela; Christiansen, Ailsa J.; Maksimow, Mikael; Varga, Zsuzsanna; Jalkanen, Sirpa; Detmar, Michael

    2016-01-01

    ABSTRACT Tumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and a strong infiltration by TAMs has been associated with estrogen receptor (ER)-negative tumors and poor prognosis. However, the molecular mechanisms behind these observations are unclear. We investigated macrophage activation in response to co-culture with several breast cancer cell lines (T47D, MCF-7, BT-474, SKBR-3, Cal-51 and MDA-MB-231) and found that high granulocyte colony-stimulating factor (G-CSF) secretion by the triple-negative breast cancer (TNBC) cell line MDA-MB-231 gave rise to immunosuppressive HLA-DRlo macrophages that promoted migration of breast cancer cells via secretion of TGF-α. In human breast cancer samples (n = 548), G-CSF was highly expressed in TNBC (p < 0.001) and associated with CD163+ macrophages (p < 0.0001), poorer overall survival (OS) (p = 0.021) and significantly increased numbers of TGF-α+ cells. While G-CSF blockade in the 4T1 mammary tumor model promoted maturation of MHCIIhi blood monocytes and TAMs and significantly reduced lung metastasis, anti-CSF-1R treatment promoted MHCIIloF4/80hiMRhi anti-inflammatory TAMs and enhanced lung metastasis in the presence of high G-CSF levels. Combined anti-G-CSF and anti-CSF-1R therapy significantly increased lymph node metastases, possibly via depletion of the so-called “gate-keeper” subcapsular sinus macrophages. These results indicate that G-CSF promotes the anti-inflammatory phenotype of tumor-induced macrophages when CSF-1R is inhibited and therefore caution against the use of M-CSF/CSF-1R targeting agents in tumors with high G-CSF expression. PMID:27141367

  14. Phenotyping studies to assess the effects of phytopharmaceuticals on in vivo activity of main human cytochrome p450 enzymes.

    PubMed

    Zadoyan, Gregor; Fuhr, Uwe

    2012-09-01

    The extensive use of herbal drugs and their multiple components and modes of action suggests that they may also cause drug interactions by changing the activity of human cytochrome P450 enzymes. The purpose of the present review is to present the available data for the top 14 herbal drug sales in the U. S. Studies describing the effects of herbal drugs on phenotyping substrates for individual CYPs were identified by a comprehensive MEDLINE search. Drugs included Allium sativum (Liliaceae), Echinacea purpurea (Asteraceae), Serenoa repens (Arecaceae), Ginkgo biloba (Ginkgoaceae), Vaccinium macrocarpon (Ericaceae), Glycine max (Fabaceae), Panax ginseng (Araliaceae), Actea racemosa (Ranunculaceae), Hypericum perforatum (Hypericaceae), Silybum marianum (Asteraceae), Camellia sinensis (Theaceae), Valeriana officinalis (Valerianaceae), Piper methysticum (Piperaceae), and Hydrastis canadensis (Ranunculaceae) preparations. We identified 70 clinical studies in 69 publications. The majority of the herbal drugs appeared to have no clear effects on most of the CYPs examined. If there was an effect, there was mild inhibition in almost all cases, as seen with garlic or kava effects on CYP2E1 and with soybean components on CYP1A2. The most pronounced effects were induction of CYP3A and other CYPs by St. John's wort and the inhibitory effect of goldenseal on CYP3A and CYP2D6, both being borderline between mild and moderate in magnitude. With the exceptions of St.John's wort and goldenseal, the information currently available suggests that concomitant intake of the herbal drugs addressed here is not a major risk for drugs that are metabolized by CYPs. PMID:22588833

  15. Theophylline Pharmacokinetics in Foetal Sheep: Maternal Metabolic Capacity is the Principal Driver.

    PubMed

    DuBois, Barent; Louey, Samantha; Giraud, George D; Cherala, Ganesh; Jonker, Sonnet S

    2015-10-01

    Understanding theophylline pharmacokinetics (PK) in the foetus is essential to prevent in utero toxicity and optimize prophylactic therapies. Previous studies in pregnancy have been obfuscated by maternal dosing and inadequate sampling in the foetus; both render modelling of foetal PK difficult. Six ewes carrying singleton foetuses received theophylline (60 mg) into the foetal jugular vein. Blood samples were drawn from the foetus and ewe over 36 hr. Serum concentrations were measured. Maternal and foetal pharmacokinetic parameters were estimated. Foetal non-compartmental pharmacokinetic parameters were as follows: half-life 7.37 ± 1.22 hr; volume of distribution 44.62 ± 11.45 L; area under the curve 14.82 ± 2.71 hr/(μg/mL); and clearance 4.15 ± 0.70 L/hr. Rapid theophylline distribution across the placenta was observed. Maternal non-compartmental pharmacokinetic parameters were as follows: half-life 6.54 ± 2.44 hr; volume of distribution 32.48 ± 9.99 L; area under the curve 16.28 ± 4.53 hr/(μg/mL); and clearance 3.69 ± 1.47 L/hr. Foetal and ewe serum concentration-time profiles were fit together into a 3-compartment population pharmacokinetic model, and parameters were as follows: central volume 1.38 ± 0.11 L; 2nd peripheral compartment volume 3.11 ± 0.29 L; 3rd peripheral compartment volume 60.14 ± 6.02 L; elimination clearance 9.89 ± 0.90 L/hr; distribution clearance between central and 2nd compartment 30.87 ± 2.31 L/hr; and distribution clearance between 2nd and 3rd compartments 13.89 ± 1.11 L/hr. Cytochrome P4501A expression was robust in maternal liver; negligible activities were observed in placenta, foetal liver and foetal kidney. In vitro protein binding of theophylline was 30% lower in foetal serum compared to maternal serum (29.7 ± 4.4 versus 42.0 ± 3.6%-bound). Free concentrations were lower in the foetus than in the ewe, suggesting active transport across placenta. In summary, foetal

  16. Common phenotypic and genotypic antimicrobial resistance patterns found in a case study of multiresistant E. coli from cohabitant pets, humans, and household surfaces.

    PubMed

    Martins, Liliana Raquel Leite; Pina, Susana Maria Rocha; Simões, Romeo Luís Rocha; de Matos, Augusto José Ferreira; Rodrigues, Pedro; da Costa, Paulo Martins Rodrigues

    2013-01-01

    The objective of the study described in this article was to characterize the antimicrobial resistance profiles among E. coli strains isolated from cohabitant pets and humans, evaluating the concurrent colonization of pets, owners, and home surfaces by bacteria carrying the same antimicrobial-resistant genes. The authors also intended to assess whether household surfaces and objects could contribute to the within-household antimicrobial-resistant gene diffusion between human and animal cohabitants. A total of 124 E. coli strains were isolated displaying 24 different phenotypic patterns with a remarkable percentage of multiresistant ones. The same resistance patterns were isolated from the dog's urine, mouth, the laundry floor, the refrigerator door, and the dog's food bowl. Some other multiresistant phenotypes, as long as resistant genes, were found repeatedly in different inhabitants and surfaces of the house. Direct, close contact between all the cohabitants and the touch of contaminated household surfaces and objects could be an explanation for these observations. PMID:23397653

  17. Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of β-cell neogenesis.

    PubMed

    Mfopou, Josué K; Houbracken, Isabelle; Wauters, Elke; Mathijs, Iris; Song, Imane; Himpe, Eddy; Baldan, Jonathan; Heimberg, Harry; Bouwens, Luc

    2016-06-01

    The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation. PMID:26987985

  18. Acinar phenotype is preserved in human exocrine pancreas cells cultured at low temperature: implications for lineage-tracing of β-cell neogenesis

    PubMed Central

    Mfopou, Josué K.; Houbracken, Isabelle; Wauters, Elke; Mathijs, Iris; Song, Imane; Himpe, Eddy; Baldan, Jonathan; Heimberg, Harry; Bouwens, Luc

    2016-01-01

    The regenerative medicine field is expanding with great successes in laboratory and preclinical settings. Pancreatic acinar cells in diabetic mice were recently converted into β-cells by treatment with ciliary neurotrophic factor (CNTF) and epidermal growth factor (EGF). This suggests that human acinar cells might become a cornerstone for diabetes cell therapy in the future, if they can also be converted into glucose-responsive insulin-producing cells. Presently, studying pancreatic acinar cell biology in vitro is limited by their high plasticity, as they rapidly lose their phenotype and spontaneously transdifferentiate to a duct-like phenotype in culture. We questioned whether human pancreatic acinar cell phenotype could be preserved in vitro by physico-chemical manipulations and whether this could be valuable in the study of β-cell neogenesis. We found that culture at low temperature (4°C) resulted in the maintenance of morphological and molecular acinar cell characteristics. Specifically, chilled acinar cells did not form the spherical clusters observed in controls (culture at 37°C), and they maintained high levels of acinar-specific transcripts and proteins. Five-day chilled acinar cells still transdifferentiated into duct-like cells upon transfer to 37°C. Moreover, adenoviral-mediated gene transfer evidenced an active Amylase promoter in the 7-day chilled acinar cells, and transduction performed in chilled conditions improved acinar cell labelling. Together, our findings indicate the maintenance of human pancreatic acinar cell phenotype at low temperature and the possibility to efficiently label acinar cells, which opens new perspectives for the study of human acinar-to-β-cell transdifferentiation. PMID:26987985

  19. Correlation between the presence of tRNA His GUG and the erythropoietic function in foetal sheep liver.

    PubMed Central

    Landin, R M; Boisnard, M; Petrissant, G

    1979-01-01

    Histidyl-tRNAs from foetal and adult sheep liver were compared to their reticulocyte counterparts. The combination of various techniques revealed the existence of two histidyl-tRNA species in reticulocytes, one of which was not retained on acetylated DBAE-cellulose columns and was guanylatable. Three histidyl-tRNA isoacceptors were identified in foetal liver. Two of these species were not adsorbed on acetylated DBAE-cellulose but only one was found to be guanylatable. An identical chromatographic behaviour on RPC-5 columns was observed for guanylated histidyl-tRNAs from both origins. These results suggest the occurrence of a GUG anticodon in these guanine-accepting tRNAs. In foetal liver the amount of guanylatable histidyl-tRNA was estimated to be 7% of the total tRNA population. This observation is in agreement with the erythropoietic function of liver during the foetal life. Images PMID:503863

  20. Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

    PubMed Central

    Shibanuma, M; Mochizuki, E; Maniwa, R; Mashimo, J; Nishiya, N; Imai, S; Takano, T; Oshimura, M; Nose, K

    1997-01-01

    The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence. PMID:9032249

  1. The human tissue-biomaterial interface: a role for PPARγ-dependent glucocorticoid receptor activation in regulating the CD163+ M2 macrophage phenotype.

    PubMed

    Bullers, Samuel J; Baker, Simon C; Ingham, Eileen; Southgate, Jennifer

    2014-09-01

    In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue-biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68(+) CD80(-) macrophages. The migration of CD68(+) cells from the tissue to the interface was accompanied by maturation to a CD163(hi) phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80(+)) toward an M2 (CD163(+)) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80(+) phenotype on glass to a CD80(-) phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163(+) phenotype, with

  2. The Human Tissue–Biomaterial Interface: A Role for PPARγ-Dependent Glucocorticoid Receptor Activation in Regulating the CD163+ M2 Macrophage Phenotype

    PubMed Central

    Bullers, Samuel J.; Baker, Simon C.; Ingham, Eileen

    2014-01-01

    In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue–biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68+ CD80− macrophages. The migration of CD68+ cells from the tissue to the interface was accompanied by maturation to a CD163hi phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80+) toward an M2 (CD163+) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80+ phenotype on glass to a CD80− phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163+ phenotype, with activation of

  3. Human Chorionic Gonadotropin (hCG) Prevents the Transformed Phenotypes Induced by 17 β-estradiol in Human Breast Epithelial Cells

    PubMed Central

    Kocdor, Hilal; Kocdor, Mehmet A.; Russo, Jose; Snider, Kara E.; Vanegas, Johana E.; Russo, Irma H.; Fernandez, Sandra V.

    2009-01-01

    Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules, We have shown that 17 β-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p<0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p<0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland. PMID:19647089

  4. siRNA Knockdown of Ribosomal Protein Gene RPL19 Abrogates the Aggressive Phenotype of Human Prostate Cancer

    PubMed Central

    Bee, Alix; Brewer, Daniel; Beesley, Carol; Dodson, Andrew; Forootan, Shiva; Dickinson, Timothy; Gerard, Patricia; Lane, Brian; Yao, Sheng; Cooper, Colin S.; Djamgoz, Mustafa B. A.; Gosden, Christine M.; Ke, Youqiang; Foster, Christopher S.

    2011-01-01

    We provide novel functional data that posttranscriptional silencing of gene RPL19 using RNAi not only abrogates the malignant phenotype of PC-3M prostate cancer cells but is selective with respect to transcription and translation of other genes. Reducing RPL19 transcription modulates a subset of genes, evidenced by gene expression array analysis and Western blotting, but does not compromise cell proliferation or apoptosis in-vitro. However, growth of xenografted tumors containing the knocked-down RPL19 in-vivo is significantly reduced. Analysis of the modulated genes reveals induction of the non-malignant phenotype principally to involve perturbation of networks of transcription factors and cellular adhesion genes. The data provide evidence that extra-ribosomal regulatory functions of RPL19, beyond protein synthesis, are critical regulators of cellular phenotype. Targeting key members of affected networks identified by gene expression analysis raises the possibility of therapeutically stabilizing a benign phenotype generated by modulating the expression of an individual gene and thereafter constraining a malignant phenotype while leaving non-malignant tissues unaffected. PMID:21799931

  5. A comparison of foetal SAR in three sets of pregnant female models.

    PubMed

    Dimbylow, Peter J; Nagaoka, Tomoaki; Xu, X George

    2009-05-01

    This paper compares the foetal SAR in the HPA hybrid mathematical phantoms with the 26-week foetal model developed at the National Institute of Information and Communications Technology, Tokyo, and the set of 13-, 26- and 38-week boundary representation models produced at Rensselaer Polytechnic Institute. FDTD calculations are performed at a resolution of 2 mm for a plane wave with a vertically aligned electric field incident upon the body from the front, back and two sides from 20 MHz to 3 GHz under isolated conditions. The external electric field values required to produce the ICNIRP public exposure localized restriction of 2 W kg(-1) when averaged over 10 g of the foetus are compared with the ICNIRP reference levels. PMID:19369706

  6. Constitutive expression of cytochrome P450 in foetal and adult porcine livers-Effects of body weight.

    PubMed

    Rasmussen, Martin Krøyer; Theil, Peter Kappel; Oksbjerg, Niels

    2016-09-01

    The liver hosts a great number of enzymatically driven processes, including detoxification. The super-family of enzymes named cytochrome P450 (CYP) is the major participant in that process. The expression of CYPs is affected by several factors including life-stage (foetal vs. adult). In the present study we investigated the impact of birth-weight (high or low birth weight) and life-stage on constitutive expression of porcine hepatic CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2C33, CYP2D25, CYP2E1 and CYP3A29, as well as the transcription factors controlling their expression; aryl hydrocarbon receptor, constitutive androstane receptor, pregnane X receptor, C/EBP and hepatocyte nuclear factors 1 and 4. Both RT-PCR and western blotting showed a marked increase in the expression of the adult pigs compared with prenatal pigs. Moreover, CYP2E1 mRNA expression was 7.5 fold higher in foetuses with low birth weight compared with foetuses with high birth weight. Gender did not affect the mRNA expression within the different life-stages. These results indicate a similarity to what is observed in humans and porcine foetuses may therefore be a model for humans when studying expression of CYPs. PMID:27320961

  7. A practical guide to non-invasive foetal electrocardiogram extraction and analysis.

    PubMed

    Behar, Joachim; Andreotti, Fernando; Zaunseder, Sebastian; Oster, Julien; Clifford, Gari D

    2016-05-01

    Non-Invasive foetal electrocardiography (NI-FECG) represents an alternative foetal monitoring technique to traditional Doppler ultrasound approaches, that is non-invasive and has the potential to provide additional clinical information. However, despite the significant advances in the field of adult ECG signal processing over the past decades, the analysis of NI-FECG remains challenging and largely unexplored. This is mainly due to the relatively low signal-to-noise ratio of the FECG compared to the maternal ECG, which overlaps in both time and frequency. This article is intended to be used by researchers as a practical guide to NI-FECG signal processing, in the context of the above issues. It reviews recent advances in NI-FECG research including: publicly available databases, NI-FECG extraction techniques for foetal heart rate evaluation and morphological analysis, NI-FECG simulators and the methodology and statistics for assessing the performance of the extraction algorithms. Reference to the most recent work is given, recent findings are highlighted in the form of intermediate summaries, references to open source code and publicly available databases are provided and promising directions for future research are motivated. In particular we emphasise the need and specifications for building a new open reference database of NI-FECG signals, and the need for new algorithms to be benchmarked on the same database, employing the same evaluation statistics. Finally we motivate the need for research in NI-FECG to address morphological analysis, since this represent one of the most promising avenues for this foetal monitoring modality. PMID:27067431

  8. Foetal dose conversion coefficients for ICRP-compliant pregnant models from idealised proton exposures.

    PubMed

    Taranenko, Valery; Xu, X George

    2009-01-01

    Protection of pregnant women and their foetus against external proton irradiations poses a unique challenge. Assessment of foetal dose due to external protons in galactic cosmic rays and as secondaries generated in aircraft walls is especially important during high-altitude flights. This paper reports a set of fluence to absorbed dose conversion coefficients for the foetus and its brain for external monoenergetic proton beams of six standard configurations (the antero-posterior, the postero-anterior, the right lateral, the left lateral, the rotational and the isotropic). The pregnant female anatomical definitions at each of the three gestational periods (3, 6 and 9 months) are based on newly developed RPI-P series of models whose organ masses were matched within 1% with the International Commission on Radiological Protection reference values. Proton interactions and the transport of secondary particles were carefully simulated using the Monte Carlo N-Particle eXtended code (MCNPX) and the phantoms consisting of several million voxels at 3 mm resolution. When choosing the physics models in the MCNPX, it was found that the advanced Cascade-Exciton intranuclear cascade model showed a maximum of 9% foetal dose increase compared with the default model combination at intermediate energies below 5 GeV. Foetal dose results from this study are tabulated and compared with previously published data that were based on simplified anatomy. The comparison showed a strong dependence upon the source geometry, energy and gestation period: the dose differences are typically less than 20% for all sources except ISO where systematically 40-80% of higher doses were observed. Below 200 MeV, a larger discrepancy in dose was found due to the Bragg peak shift caused by different anatomy. The tabulated foetal doses represent the latest and most detailed study to date offering a useful set of data to improve radiation protection dosimetry against external protons. PMID:19246483

  9. Combining and benchmarking methods of foetal ECG extraction without maternal or scalp electrode data.

    PubMed

    Behar, Joachim; Oster, Julien; Clifford, Gari D

    2014-08-01

    Despite significant advances in adult clinical electrocardiography (ECG) signal processing techniques and the power of digital processors, the analysis of non-invasive foetal ECG (NI-FECG) is still in its infancy. The Physionet/Computing in Cardiology Challenge 2013 addresses some of these limitations by making a set of FECG data publicly available to the scientific community for evaluation of signal processing techniques.The abdominal ECG signals were first preprocessed with a band-pass filter in order to remove higher frequencies and baseline wander. A notch filter to remove power interferences at 50 Hz or 60 Hz was applied if required. The signals were then normalized before applying various source separation techniques to cancel the maternal ECG. These techniques included: template subtraction, principal/independent component analysis, extended Kalman filter and a combination of a subset of these methods (FUSE method). Foetal QRS detection was performed on all residuals using a Pan and Tompkins QRS detector and the residual channel with the smoothest foetal heart rate time series was selected.The FUSE algorithm performed better than all the individual methods on the training data set. On the validation and test sets, the best Challenge scores obtained were E1 = 179.44, E2 = 20.79, E3 = 153.07, E4 = 29.62 and E5 = 4.67 for events 1-5 respectively using the FUSE method. These were the best Challenge scores for E1 and E2 and third and second best Challenge scores for E3, E4 and E5 out of the 53 international teams that entered the Challenge. The results demonstrated that existing standard approaches for foetal heart rate estimation can be improved by fusing estimators together. We provide open source code to enable benchmarking for each of the standard approaches described. PMID:25069410

  10. Mechanisms of 5-azacytidine (5AzC)-induced toxicity in the rat foetal brain

    PubMed Central

    Ueno, Masaki; Katayama, Kei-Ichi; Nakayama, Hiroyuki; Doi, Kunio

    2002-01-01

    Mechanisms of 5-azacytidine (5AzC)-induced toxicity in the rat foetal brain were investigated. 5AzC (10 mg/kg) was injected into pregnant rats on day 13 of gestation and the protein and mRNA expressions of p53 and its transcriptional target genes, p21, bax, cyclin G1, fas, and gadd45, were examined in the foetal brain. The number of p53-positive cells peaked at 9 h after treatment (HAT) and those of apoptotic cells and p21-positive cells peaked at 12 HAT. The expressions of p21, bax, cyclin G1, and fas mRNAs were significantly elevated from 9 to 12 HAT. From the experiments using 5-bromo-2′-deoxyuridine (BrdU), as compared with controls, the migration of neuroepithelial cells significantly delayed and BrdU-positive signals were observed in many apoptotic cells from 9 to 24 HAT in the 5AzC-group. In addition, the number of S phase cells significantly decreased at 12 HAT. The present results indicate that 5AzC induced apoptosis and cell cycle arrest probably at G1 phase in the rat foetal brain and they might be mediated by p53 in response to DNA damage. PMID:12383193

  11. Controversies and considerations regarding the termination of pregnancy for Foetal Anomalies in Islam

    PubMed Central

    2014-01-01

    Background Approximately one-fourth of all the inhabitants on earth are Muslims. Due to unprecedented migration, physicians are often confronted with cultures other than their own that adhere to different pdigms. Discussion In Islam, and most religions, abortion is forbidden. Islam is considerably liberal concerning abortion, which is dependent on (i) the threat of harm to mothers, (ii) the status of the pregnancy before or after ensoulment (on the 120th day of gestation), and (iii) the presence of foetal anomalies that are incompatible with life. Considerable variation in religious edicts exists, but most Islamic scholars agree that the termination of a pregnancy for foetal anomalies is allowed before ensoulment, after which abortion becomes totally forbidden, even in the presence of foetal abnormalities; the exception being a risk to the mother’s life or confirmed intrauterine death. Summary The authors urge Muslim law makers to also consider abortion post ensoulment if it is certain that the malformed foetus will decease soon after birth or will be severely malformed and physically and mentally incapacitated after birth to avoid substantial hardship that may continue for years for mothers and family members. The authors recommend that an institutional committee governed and monitored by a national committee make decisions pertaining to abortion to ensure that ethics are preserved and mistakes are prevented. Anomalous foetuses must be detected at the earliest possible time to enable an appropriate medical intervention prior to the 120th day. PMID:24499356

  12. Foetal phonocardiographic signal denoising based on non-negative matrix factorization.

    PubMed

    Chourasia, V S; Tiwari, A K; Gangopadhyay, R; Akant, K A

    2012-01-01

    Foetal phonocardiography (fPCG) is a non-invasive, cost-effective and simple technique for antenatal care. The fPCG signals contain vital information of diagnostic importance regarding the foetal health. However, the fPCG signal is usually contaminated by various noises and thus requires robust signal processing to denoise the signal. The main aim of this paper is to develop a methodology for removal of unwanted noise from the fPCG signal. The proposed methodology utilizes the non-negative matrix factorization (NMF) algorithm. The developed methodology is tested on both simulated and real-time fPCG signals. The performance of the developed methodology has been evaluated in terms of the gain in signal-to-noise ratio (SNR) achieved through the process of denoising. In particular, using the NMF algorithm, a substantial improvement in SNR of the fPCG signals in the range of 12-30 dB has been achieved, providing a high quality assessment of foetal well-being. PMID:22136609

  13. Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

    PubMed Central

    Bose, Bipasha; Ordovas, Laura; Vanuytsel, Kim; Geraerts, Martine; Firpo, Meri; De Vos, Rita; Fevery, Johan; Nevens, Frederik; Hu, Wei-Shou; Verfaillie, Catherine M.

    2010-01-01

    Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development. PMID:20711405

  14. A Case of Bordetella brochiseptica at a Military Medical Facility in Hawai‘i: Phenotypic and Molecular Testing of an Uncommon Human Pathogen

    PubMed Central

    Agee, Willie A; Kajiura, Lauren; Hawley-Molloy, Joshua S; Staege, Catherine M; Barnhill, Jason C

    2015-01-01

    Bordetella bronchiseptica (B. bronchiseptica) is rarely implicated in human disease. Human infections typically occur in the context of immunosuppression and while human infection has been sporadically reported in the literature, the majority of these reports are largely descriptive and do not explore the molecular and phenotypic properties of the isolates in question. Here we report the isolation and characterization of a B. bronchiseptica isolate derived from an HIV positive patient at Tripler Army Medical Center on O‘ahu. This case represents the first published report of human infection of B. bronchiseptica in the state of Hawai‘i and the most detailed description of the biochemical and molecular features of a Hawaiian isolate to date. PMID:26225268

  15. Mammary-digital-nail (MDN) syndrome: a novel phenotype maps to human chromosome 22q12.3–13.1

    PubMed Central

    Genzer-Nir, Mira; Khayat, Morad; Kogan, Leonid; Cohen, Hector I; Hershkowitz, Miriam; Geiger, Dan; Falik-Zaccai, Tzipora C

    2010-01-01

    Mammary-digital-nail syndrome is a novel phenotypic association consisting of anonychia onychodystrophy with hypoplasia or absence of distal phalanges in males and females, accompanied by juvenile hypertrophy of the breast in affected females. This newly described genetic trait presents an autosomal dominant inheritance pattern, with either reduced penetrance or germ-line mosaicism. Analysis of the pedigree, linkage studies followed by a genome-wide screen and by haplotype analysis defined the locus for the phenotype within a 12 cM (4.3 Mb) interval on chromosome 22q12.3-13.1. This chromosomal region has not been implicated before in genetic disorders of the mammary tissue or limbs. These data suggest a possibly novel signaling pathway affecting the organogenesis of limbs and mammary glands in humans. PMID:20145678

  16. Simultaneous suppression of epidermal growth factor receptor and c-erbB-2 reverses aneuploidy and malignant phenotype of a human ovarian carcinoma cell line.

    PubMed

    Pack, Svetlana D; Alper, Ozgül M; Stromberg, Kurt; Augustus, Meena; Ozdemirli, Metin; Miermont, Anne M; Klus, Greg; Rusin, Marek; Slack, Rebecca; Hacker, Neville F; Ried, Thomas; Szallasi, Zoltan; Alper, Ozge

    2004-02-01

    Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47-68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype. PMID:14871800

  17. Concordant but Varied Phenotypes among Duchenne Muscular Dystrophy Patient-Specific Myoblasts Derived using a Human iPSC-Based Model.

    PubMed

    Choi, In Young; Lim, HoTae; Estrellas, Kenneth; Mula, Jyothi; Cohen, Tatiana V; Zhang, Yuanfan; Donnelly, Christopher J; Richard, Jean-Philippe; Kim, Yong Jun; Kim, Hyesoo; Kazuki, Yasuhiro; Oshimura, Mitsuo; Li, Hongmei Lisa; Hotta, Akitsu; Rothstein, Jeffrey; Maragakis, Nicholas; Wagner, Kathryn R; Lee, Gabsang

    2016-06-01

    Duchenne muscular dystrophy (DMD) remains an intractable genetic disease. Althogh there are several animal models of DMD, there is no human cell model that carries patient-specific DYSTROPHIN mutations. Here, we present a human DMD model using human induced pluripotent stem cells (hiPSCs). Our model reveals concordant disease-related phenotypes with patient-dependent variation, which are partially reversed by genetic and pharmacological approaches. Our "chemical-compound-based" strategy successfully directs hiPSCs into expandable myoblasts, which exhibit a myogenic transcriptional program, forming striated contractile myofibers and participating in muscle regeneration in vivo. DMD-hiPSC-derived myoblasts show disease-related phenotypes with patient-to-patient variability, including aberrant expression of inflammation or immune-response genes and collagens, increased BMP/TGFβ signaling, and reduced fusion competence. Furthermore, by genetic correction and pharmacological "dual-SMAD" inhibition, the DMD-hiPSC-derived myoblasts and genetically corrected isogenic myoblasts form "rescued" multi-nucleated myotubes. In conclusion, our findings demonstrate the feasibility of establishing a human "DMD-in-a-dish" model using hiPSC-based disease modeling. PMID:27239027

  18. Overexpression of Snail induces epithelial–mesenchymal transition and a cancer stem cell–like phenotype in human colorectal cancer cells

    PubMed Central

    Fan, Fan; Samuel, Shaija; Evans, Kurt W; Lu, Jia; Xia, Ling; Zhou, Yunfei; Sceusi, Eric; Tozzi, Federico; Ye, Xiang-Cang; Mani, Sendurai A; Ellis, Lee M

    2012-01-01

    Epithelial–mesenchymal transition (EMT) is a critical process providing tumor cells with the ability to migrate and escape from the primary tumor and metastasize to distant sites. Recently, EMT was shown to be associated with the cancer stem cell (CSC) phenotype in breast cancer. Snail is a transcription factor that mediates EMT in a number of tumor types, including colorectal cancer (CRC). Our study was done to determine the role of Snail in mediating EMT and CSC function in CRC. Human CRC specimens were stained for Snail expression, and human CRC cell lines were transduced with a retroviral Snail construct or vector control. Cell proliferation and chemosensitivity to oxaliplatin of the infected cells were determined by the MTT (colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Migration and invasion were determined in vitro using modified Boyden chamber assays. EMT and putative CSC markers were analyzed using Western blotting. Intravenous injection of tumor cells was done to evaluate their metastatic potential in mice. Snail was overexpressed in human CRC surgical specimens. This overexpression induced EMT and a CSC-like phenotype in human CRC cells and enhanced cell migration and invasion (P < 0.002 vs. control). Snail overexpression also led to an increase in metastasis formation in vivo (P < 0.002 vs. control). Furthermore, the Snail-overexpressing CRC cells were more chemoresistant to oxaliplatin than control cells. Increased Snail expression induces EMT and the CSC-like phenotype in CRC cells, which enhance cancer cell invasion and chemoresistance. Thus, Snail is a potential therapeutic target in metastatic CRC. PMID:23342249

  19. Bioimaging for quantitative phenotype analysis.

    PubMed

    Chen, Weiyang; Xia, Xian; Huang, Yi; Chen, Xingwei; Han, Jing-Dong J

    2016-06-01

    With the development of bio-imaging techniques, an increasing number of studies apply these techniques to generate a myriad of image data. Its applications range from quantification of cellular, tissue, organismal and behavioral phenotypes of model organisms, to human facial phenotypes. The bio-imaging approaches to automatically detect, quantify, and profile phenotypic changes related to specific biological questions open new doors to studying phenotype-genotype associations and to precisely evaluating molecular changes associated with quantitative phenotypes. Here, we review major applications of bioimage-based quantitative phenotype analysis. Specifically, we describe the biological questions and experimental needs addressable by these analyses, computational techniques and tools that are available in these contexts, and the new perspectives on phenotype-genotype association uncovered by such analyses. PMID:26850283

  20. Toward harmonized phenotyping of human myeloid-derived suppressor cells by flow cytometry: results from an interim study.

    PubMed

    Mandruzzato, Susanna; Brandau, Sven; Britten, Cedrik M; Bronte, Vincenzo; Damuzzo, Vera; Gouttefangeas, Cécile; Maurer, Dominik; Ottensmeier, Christian; van der Burg, Sjoerd H; Welters, Marij J P; Walter, Steffen

    2016-02-01

    There is an increasing interest for monitoring circulating myeloid-derived suppressor cells (MDSCs) in cancer patients, but there are also divergences in their phenotypic definition. To overcome this obstacle, the Cancer Immunoguiding Program under the umbrella of the Association of Cancer Immunotherapy is coordinating a proficiency panel program that aims at harmonizing MDSC phenotyping. After a consultation period, a two-stage approach was designed to harmonize MDSC phenotype. In the first step, an international consortium of 23 laboratories immunophenotyped 10 putative MDSC subsets on pretested, peripheral blood mononuclear cells of healthy donors to assess the level of concordance and define robust marker combinations for the identification of circulating MDSCs. At this stage, no mandatory requirements to standardize reagents or protocols were introduced. Data analysis revealed a small intra-laboratory, but very high inter-laboratory variance for all MDSC subsets, especially for the granulocytic subsets. In particular, the use of a dead-cell marker altered significantly the reported percentage of granulocytic MDSCs, confirming that these cells are especially sensitive to cryopreservation and/or thawing. Importantly, the gating strategy was heterogeneous and associated with high inter-center variance. Overall, our results document the high variability in MDSC phenotyping in the multicenter setting if no harmonization/standardization measures are applied. Although the observed variability depended on a number of identified parameters, the main parameter associated with variation was the gating strategy. Based on these findings, we propose further efforts to harmonize marker combinations and gating parameters to identify strategies for a robust enumeration of MDSC subsets. PMID:26728481

  1. Nonsense mutations in the rhodopsin gene that give rise to mild phenotypes trigger mRNA degradation in human cells by nonsense-mediated decay.

    PubMed

    Roman-Sanchez, Ramon; Wensel, Theodore G; Wilson, John H

    2016-04-01

    Eight different nonsense mutations in the human rhodopsin gene cause retinitis pigmentosa (RP), an inherited degenerative disease of the retina that can lead to complete blindness. Although all these nonsense mutations lead to premature termination codons (PTCs) in rhodopsin mRNA, some display dominant inheritance, while others are recessive. Because nonsense-mediated decay (NMD) can degrade mRNAs containing PTCs and modulate the inheritance patterns of genetic diseases, we asked whether any of the nonsense mutations in the rhodopsin gene generated mRNAs that were susceptible to degradation by NMD. We hypothesized that nonsense mutations that caused mild RP phenotypes would trigger NMD, whereas those that did not engage NMD would cause more severe RP phenotypes-presumably due to the toxicity of the truncated protein. To test our hypothesis, we transfected human rhodopsin nonsense mutants into HEK293 and HT1080 human cells and measured transcript levels by qRT-PCR. In both cell lines, rhodopsin mutations Q64X and Q344X, which cause severe phenotypes that are dominantly inherited, yielded the same levels of rhodopsin mRNA as wild type. By contrast, rhodopsin mutations W161X and E249X, which cause recessive RP, showed decreased rhodopsin mRNA levels, consistent with NMD. Rhodopsin mutant Y136X, a dominant mutation that causes late-onset RP with a very mild pathology, also gave lower mRNA levels. Treatment of cells with Wortmannin, an inhibitor of NMD, eliminated the degradation of Y136X, W161X, and E249X rhodopsin mRNAs. These results suggest that NMD modulates the severity of RP in patients with nonsense mutations in the rhodopsin gene. PMID:26416182

  2. Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation

    PubMed Central

    Filograna, Roberta; Civiero, Laura; Ferrari, Vanni; Codolo, Gaia; Greggio, Elisa; Bubacco, Luigi; Beltramini, Mariano; Bisaglia, Marco

    2015-01-01

    Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field. PMID:26317353

  3. Targeting ID2 expression triggers a more differentiated phenotype and reduces aggressiveness in human salivary gland cancer cells.

    PubMed

    Sumida, Tomoki; Ishikawa, Akiko; Nakano, Hiroyuki; Yamada, Tomohiro; Mori, Yoshihide; Desprez, Pierre-Yves

    2016-08-01

    Inhibitors of DNA-binding (ID) proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. We previously determined that ID1 was highly expressed in aggressive salivary gland cancer (SGC) cells in culture. Here, we show that ID2 is also expressed in aggressive SGC cells. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. Moreover, ID2 knockdown almost completely suppresses invasion and the expression of matrix metalloproteinase 9. In conclusion, ID2 expression maintains an aggressive phenotype in SGC cells, and ID2 repression triggers a reduction in cell aggressiveness. ID2 therefore represents a potential therapeutic target during SGC progression. ID proteins are negative regulators of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. ID2 knockdown triggers important changes in cell behavior, that is, it significantly reduces the expression of N-cadherin, vimentin and Snail, induces E-cadherin expression and leads to a more differentiated phenotype exemplified by changes in cell shape. ID2 therefore represents a potential therapeutic target during SGC progression. PMID:27364596

  4. Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

    PubMed

    Mhashilkar, Amruta S; Adapa, Swamy R; Jiang, Rays H Y; Williams, Steven A; Zaky, Weam; Slatko, Barton E; Luck, Ashley N; Moorhead, Andrew R; Unnasch, Thomas R

    2016-05-01

    A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20-hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20-hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development. PMID:26896576

  5. Altered GPM6A/M6 dosage impairs cognition and causes phenotypes responsive to cholesterol in human and Drosophila.

    PubMed

    Gregor, Anne; Kramer, Jamie M; van der Voet, Monique; Schanze, Ina; Uebe, Steffen; Donders, Rogier; Reis, André; Schenck, Annette; Zweier, Christiane

    2014-12-01

    Glycoprotein M6A (GPM6A) is a neuronal transmembrane protein of the PLP/DM20 (proteolipid protein) family that associates with cholesterol-rich lipid rafts and promotes filopodia formation. We identified a de novo duplication of the GPM6A gene in a patient with learning disability and behavioral anomalies. Expression analysis in blood lymphocytes showed increased GPM6A levels. An increase of patient-derived lymphoblastoid cells carrying membrane protrusions supports a functional effect of this duplication. To study the consequences of GPM6A dosage alterations in an intact nervous system, we employed Drosophila melanogaster as a model organism. We found that knockdown of Drosophila M6, the sole member of the PLP family in flies, in the wing, and whole organism causes malformation and lethality, respectively. These phenotypes as well as the protrusions of patient-derived lymphoblastoid cells with increased GPM6A levels can be alleviated by cholesterol supplementation. Notably, overexpression as well as loss of M6 in neurons specifically compromises long-term memory in the courtship conditioning paradigm. Our findings thus indicate a critical role of correct GPM6A/M6 levels for cognitive function and support a role of the GPM6A duplication for the patient's phenotype. Together with other recent findings, this study highlights compromised cholesterol homeostasis as a recurrent feature in cognitive phenotypes. PMID:25224183

  6. Functional and Phenotypic Characteristics of Alternative Activation Induced in Human Monocytes by Interleukin-4 or the Parasitic Nematode Brugia malayi ▿ †

    PubMed Central

    Semnani, Roshanak Tolouei; Mahapatra, Lily; Moore, Vanessa; Sanprasert, Vivornpun; Nutman, Thomas B.

    2011-01-01

    Human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (MΦ). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 [IL-4]) or classically (macrophage colony-stimulating factor [MCSF]) activated MΦ. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of MΦ but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4+ and CD8+ T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. In contrast to results with MCSF-cultured monocytes, exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly. PMID:21788379

  7. Phenotypic Characterization of Mice Carrying Homozygous Deletion of KLF11, a Gene in Which Mutations Cause Human Neonatal and MODY VII Diabetes.

    PubMed

    Mathison, Angela; Escande, Carlos; Calvo, Ezequiel; Seo, Seungmae; White, Thomas; Salmonson, Ann; Faubion, William A; Buttar, Navtej; Iovanna, Juan; Lomberk, Gwen; Chini, Eduardo N; Urrutia, Raul

    2015-10-01

    We have previously shown that amino acid changes in the human Kruppel-Like Factor (KLF) 11 protein is associated with the development of maturity onset diabetes of the young VII, whereas complete inactivation of this pathway by the -331 human insulin mutation causes neonatal diabetes mellitus. Here, we report that Klf11-/- mice have decreased circulating insulin levels, alterations in the control of blood glucose and body weight, as well as serum dyslipidemia, but do not develop diabetes. Functional assays using ex vivo liver tissue sections demonstrate that Klf11-/- mice display increased insulin sensitivity. Genome-wide experiments validated by pathway-specific quantitative PCR arrays reveal that the Klf11-/- phenotype associates to alterations in the regulation of gene networks involved in lipid metabolism, in particular those regulated by peroxisome proliferator-activated receptor-γ. Combined, these results demonstrate that the major phenotype given by the whole-body deletion of Klf11 in mouse is not diabetes but increased insulin sensitivity, likely due to altered transcriptional regulation in target tissues. The absence of diabetes in the Klf11-/- mouse either indicates an interspecies difference for the role of this transcription factor in metabolic homeostasis between mouse and humans, or potentially highlights the fact that other molecular factors can compensate for its absence. Nevertheless, the data of this study, gathered at the whole-organism level, further support a role for KLF11 in metabolic processes like insulin sensitivity, which regulation is critical in several forms of diabetes. PMID:26248217

  8. Local immunosuppression induced by high viral load of human papillomavirus: characterization of cellular phenotypes producing interleukin-10 in cervical neoplastic lesions.

    PubMed

    Prata, Thiago Theodoro Martins; Bonin, Camila Mareti; Ferreira, Alda Maria Teixeira; Padovani, Cacilda Tezelli Junqueira; Fernandes, Carlos Eurico dos Santos; Machado, Ana Paula; Tozetti, Inês Aparecida

    2015-09-01

    A specific immune response to human papillomavirus (HPV) in the cervical microenvironment plays a key role in eradicating infection and eliminating mutated cells. However, high-risk HPVs modulate immune cells to create an immunosuppressive microenvironment, and induce these immune cells to produce interleukin 10 (IL-10). This production of IL-10, in conjunction with HPV infection, contributes to the appearance of cervical neoplastic lesions. We sought to characterize the IL-10-producing cellular phenotype, and investigate the influence of host and HPV factors upon the induction of an immunosuppressive microenvironment. Immunohistochemical analysis demonstrated an increase in IL-10 production by keratinocytes, macrophages and Langerhans cells in high-grade cervical lesions and cervical cancer. This increase was more pronounced in patients older than 30 years, and was also correlated with high viral load, and infection with a single HPV type, particularly high-risk HPVs. Our results indicate the existence of a highly immunosuppressive microenvironment composed of different IL-10-producing cellular phenotypes in cervical cancer samples, and samples classified as high-grade cervical lesions (cervical intraepithelial neoplasia stages II and III). The immunosuppressive microenvironment that developed for these different cellular phenotypes favours viral persistence and neoplastic progression. PMID:26059395

  9. Variability of the Phenotype and Proliferation and Migration Characteristics of Human Mesenchymal Stromal Cells Derived from the Deciduous Teeth Pulp of Different Donors.

    PubMed

    Vakhrushev, I V; Vdovin, A S; Strukova, L A; Yarygin, K N

    2016-02-01

    We performed a comparative study of cell phenotype and proliferation and migration activities in vitro of mesenchymal stromal cells from human exfoliated deciduous teeth (SHED cells) from three donors. In the primary cultures, the cells of different donors had the same morphology and cytophenotype, but differed by proliferative and migration capacities. The results indicate that individual mesenchymal stromal cells cultures can differ considerably by important cell properties, and this should be considered when evaluating their potential therapeutic efficacy and in experimental studies. PMID:26899847

  10. Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents.

    PubMed

    Müssig, Eva; Steinberg, Thorsten; Kohl, Annette; Chamulitrat, Walee; Komposch, Gerda; Tomakidi, Pascal

    2008-04-01

    Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2 weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits alpha6 and beta1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of beta1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin alpha6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and alpha6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation. PMID:18188601

  11. Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

    PubMed Central

    Andolfi, Grazia; Fousteri, Georgia; Rossetti, Maura; Magnani, Chiara F; Jofra, Tatiana; Locafaro, Grazia; Bondanza, Attilio; Gregori, Silvia; Roncarolo, Maria-Grazia

    2012-01-01

    Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10–producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10–transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells. PMID:22692497

  12. A Non Leaky Artemis-Deficient Mouse that Accurately Models the Human SCID Phenotype Including Resistance to Hematopoietic Stem Cell Transplantation

    PubMed Central

    Xiao, Zheng; Dunn, Elizabeth; Singh, Kanal; Khan, Imran S.; Yannone, Steven M.; Cowan, Morton J.

    2009-01-01

    Two Artemis-deficient (mArt-/-) mouse models, independently generated on 129/SvJ backgrounds have the expected T-B-NK+SCID phenotype. However, they fail to mimic the human disease due to CD4+ T-cell leakiness. Moreover, immune reconstitution in these leaky mouse models following hematopoietic stem cell transplantation (HSCT) is more easily achieved than that seen in Artemis-deficient humans. To develop a more clinically relevant animal model we backcrossed the mArt-/- mutation onto the C57Bl/6 (B6) background (99.9%) resulting in virtually no CD4+ T-cell leakiness compared to 129/SvJ mArt-/- mice (0.3±0.25% vs 19.5±15.1%, p<0.001). The non-leaky mouse was also uniquely resistant to engraftment using allogeneic mismatched HSC, comparable to what is seen with human Artemis deficiency. The genetic background also influenced Artemis-associated radiation sensitivity with differing degrees of x-ray hypersensitivity evident in 129/SvJ and B6 backgrounds with both the mArt-/- and mArt-/+ genotypes. Our results indicate that immunogenic and DNA repair phenotypes associated with Artemis deficiency are significantly altered by genetic background, which has important implications for SCID diagnosis and treatment. Moreover, the B6 mArt-/- mouse is a more accurate model for the human disease, and a more appropriate system for studying human Artemis-deficiency and for developing improved transplant and gene therapy regimens for the treatment of SCID children. PMID:19135937

  13. Phenotypic, Genotypic, and Antimicrobial Characteristics of Streptococcus halichoeri Isolates from Humans, Proposal To Rename Streptococcus halichoeri as Streptococcus halichoeri subsp. halichoeri, and Description of Streptococcus halichoeri subsp. hominis subsp. nov., a Bacterium Associated with Human Clinical Infections.

    PubMed

    Shewmaker, P L; Whitney, A M; Humrighouse, B W

    2016-03-01

    Phenotypic, genotypic, and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA, and recN genes; comparative whole-genome analysis; conventional biochemical and Rapid ID 32 Strep identification methods; and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA, and recN gene s